Compositions and methods for modulating SMN gene family expression

ABSTRACT

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of SMN1 or SMN2. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of SMN1 or SMN2 that comprises exon 7. Methods for modulating expression of SMN1 or SMN2 using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of SMN1 or SMN2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part under 35 U.S.C. 111(a) ofU.S. patent application Ser. No. 14/401,194, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING SMN GENE FAMILY EXPRESSION”, filed on Nov. 14,2014, which is a national stage filing under U.S.C. § 371 of PCTInternational Application PCT/US2013/041440, with an internationalfiling date of May 16, 2013, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/785,529, entitled“COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION”,filed Mar. 14, 2013; U.S. Provisional Application No. 61/719,394,entitled “COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILYEXPRESSION”, filed Oct. 27, 2012; and U.S. Provisional Application No.61/647,858, entitled “COMPOSITIONS AND METHODS FOR MODULATING SMN GENEFAMILY EXPRESSION”, filed May 16, 2012, and is a continuation-in part ofU.S. patent application Ser. No. 14/401,196, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING UTRN EXPRESSION”, filed on Nov. 14, 2014, whichis a national stage filing under U.S.C. § 371 of PCT InternationalApplication PCT/US2013/041452, with an international filing date of May16, 2013, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/647,886, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING UTRN EXPRESSION”, filed May 16, 2012, and is acontinuation-in part of U.S. patent application Ser. No. 14/401,201,entitled “COMPOSITIONS AND METHODS FOR MODULATING HEMOGLOBIN GENE FAMILYEXPRESSION”, filed on Nov. 14, 2014, which is a national stage filingunder U.S.C. § 371 of PCT International Application PCT/US2013/041382,with an international filing date of May 16, 2013, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.61/785,956, entitled “COMPOSITIONS AND METHODS FOR MODULATING HEMOGLOBINGENE FAMILY EXPRESSION”, filed Mar. 14, 2013 and U.S. ProvisionalApplication No. 61/647,901, entitled “COMPOSITIONS AND METHODS FORMODULATING HEMOGLOBIN GENE FAMILY EXPRESSION”, filed May 16, 2012, andis a continuation-in part of U.S. patent application Ser. No.14/401,214, entitled “COMPOSITIONS AND METHODS FOR MODULATING ATP2A2EXPRESSION”, filed on Nov. 14, 2014, which is a national stage filingunder U.S.C. § 371 of PCT International Application PCT/US2013/041381,with an international filing date of May 16, 2013, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.61/785,832, entitled “COMPOSITIONS AND METHODS FOR MODULATING ATP2A2EXPRESSION”, filed Mar. 14, 2013 and U.S. Provisional Application No.61/647,925, entitled “COMPOSITIONS AND METHODS FOR MODULATING ATP2A2EXPRESSION”, filed May 16, 2012, and is a continuation-in part of U.S.patent application Ser. No. 14/401,223, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING APOA1 AND ABCA1 EXPRESSION”, filed on Nov. 14,2014, which is a national stage filing under U.S.C. § 371 of PCTInternational Application PCT/US2013/041455, with an internationalfiling date of May 16, 2013, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/785,778, entitled“COMPOSITIONS AND METHODS FOR MODULATING APOA1 AND ABCA1 EXPRESSION”,filed Mar. 14, 2013 and U.S. Provisional Application No. 61/647,949,entitled “COMPOSITIONS AND METHODS FOR MODULATING APOA1 AND ABCA1EXPRESSION”, filed May 16, 2012, and is a continuation-in part of U.S.patent application Ser. No. 14/401,227, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING PTEN EXPRESSION”, filed on Nov. 14, 2014, whichis a national stage filing under U.S.C. § 371 of PCT InternationalApplication PCT/US2013/041389, with an international filing date of May16, 2013, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/785,885, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING PTEN EXPRESSION”, filed Mar. 14, 2013 and U.S.Provisional Application No. 61/648,041, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING PTEN EXPRESSION”, filed May 16, 2012, and is acontinuation-in part of U.S. patent application Ser. No. 14/401,234,entitled “COMPOSITIONS AND METHODS FOR MODULATING BDNF EXPRESSION”,filed on Nov. 14, 2014, which is a national stage filing under U.S.C. §371 of PCT International Application PCT/US2013/041385, with aninternational filing date of May 16, 2013, which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/648,058,entitled “COMPOSITIONS AND METHODS FOR MODULATING BDNF EXPRESSION”,filed May 16, 2012, and is a continuation-in part of U.S. patentapplication Ser. No. 15/787,876, entitled “COMPOSITIONS AND METHODS FORMODULATING MECP2 EXPRESSION”, filed on Oct. 19, 2017, which is acontinuation of U.S. patent application Ser. No. 14/401,237, entitled“COMPOSITIONS AND METHODS FOR MODULATING MECP2 EXPRESSION”, filed onNov. 14, 2014, which is a national stage filing under U.S.C. § 371 ofPCT International Application PCT/US2013/041394, with an internationalfiling date of May 16, 2013, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/648,051, entitled“COMPOSITIONS AND METHODS FOR MODULATING MECP2 EXPRESSION”, filed May16, 2012, and is a continuation-in part of U.S. patent application Ser.No. 14/401,240, entitled “COMPOSITIONS AND METHODS FOR MODULATING GENEEXPRESSION”, filed on Nov. 14, 2014, which is a national stage filingunder U.S.C. § 371 of PCT International Application PCT/US2013/041434,with an international filed date of May 16, 2013, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.61/647,915, entitled “COMPOSITIONS AND METHODS FOR MODULATING CFTREXPRESSION”, filed May 16, 2012; U.S. Provisional Application No.61/647,938, entitled “COMPOSITIONS AND METHODS FOR MODULATING PAHEXPRESSION”, filed May 16, 2012; U.S. Provisional Application No.61/648,030, entitled “COMPOSITIONS AND METHODS FOR MODULATING CEP290EXPRESSION”, filed May 16, 2012; U.S. Provisional Application No.61/648,045, entitled “COMPOSITIONS AND METHODS FOR MODULATING ADIPOQEXPRESSION”, filed May 16, 2012; U.S. Provisional Application No.61/648,052, entitled “COMPOSITIONS AND METHODS FOR MODULATING CD274EXPRESSION”, filed May 16, 2012; U.S. Provisional Application No.61/648,069, entitled “COMPOSITIONS AND METHODS FOR MODULATING GENEEXPRESSION”, filed May 16, 2012; U.S. Provisional Application No.61/786,095, entitled “COMPOSITIONS AND METHODS FOR MODULATING GENEEXPRESSION”, filed Mar. 14, 2013, and is a continuation-in part of U.S.patent application Ser. No. 14/401,248, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING GENE EXPRESSION”, filed on Nov. 14, 2014, whichis a national stage filing under U.S.C. § 371 of PCT InternationalApplication PCT/US2013/041437, with an international filing date of May16, 2013, which, claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/648,077, entitled, “COMPOSITIONS ANDMETHODS FOR MODULATING GENE EXPRESSION”, filed on May 16, 2012, and is acontinuation-in part of U.S. patent application Ser. No. 14/691,361entitled “COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION”,filed on Apr. 20, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/401,252, entitled “COMPOSITIONS AND METHODS FORMODULATING GENE EXPRESSION”, filed on Nov. 14, 2014, which is a nationalstage filing under U.S.C. § 371 of PCT International ApplicationPCT/US2013/041461, with an international filing date of May 16, 2013,which claims the benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/648,016, entitled, “COMPOSITIONS AND METHODS FORMODULATING GENE EXPRESSION”, filed on May 16, 2012, of U.S. ProvisionalApplication No. 61/648,021, entitled, “COMPOSITIONS AND METHODS FORMODULATING GENE EXPRESSION”, filed on May 16, 2012, of U.S. ProvisionalApplication No. 61/786,232, entitled, “COMPOSITIONS AND METHODS FORMODULATING GENE EXPRESSION”, filed on Mar. 14, 2013, of U.S. ProvisionalApplication No. 61/647,858, entitled, “COMPOSITIONS AND METHODS FORMODULATING SMN GENE FAMILY EXPRESSION”, filed on May 16, 2012, of U.S.Provisional Application No. 61/719,394, entitled, “COMPOSITIONS ANDMETHODS FOR MODULATING SMN GENE FAMILY EXPRESSION”, filed on Oct. 27,2012, of U.S. Provisional Application No. 61/785,529, entitled,“COMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION”,filed on Mar. 14, 2013, of U.S. Provisional Application No. 61/647,886,entitled, “COMPOSITIONS AND METHODS FOR MODULATING UTRN EXPRESSION”,filed on May 16, 2012, of U.S. Provisional Application No. 61/647,901,entitled, “COMPOSITIONS AND METHODS FOR MODULATING HEMOGLOBIN GENEFAMILY EXPRESSION”, filed on May 16, 2012, of U.S. ProvisionalApplication No. 61/785,956, entitled, “COMPOSITIONS AND METHODS FORMODULATING HEMOGLOBIN GENE FAMILY EXPRESSION”, filed on Mar. 14, 2013,of U.S. Provisional Application No. 61/647,925, entitled, “COMPOSITIONSAND METHODS FOR MODULATING ATP2A2 EXPRESSION”, filed on May 16, 2012, ofU.S. Provisional Application No. 61/785,832, entitled, “COMPOSITIONS ANDMETHODS FOR MODULATING ATP2A2 EXPRESSION”, filed on Mar. 14, 2013, ofU.S. Provisional Application No. 61/647,949, entitled, “COMPOSITIONS ANDMETHODS FOR MODULATING APOA1 AND ABCA1 EXPRESSION”, filed on May 16,2012, of U.S. Provisional Application No. 61/785,778, entitled,“COMPOSITIONS AND METHODS FOR MODULATING APOA1 AND ABCA1 EXPRESSION”,filed on Mar. 14, 2013, of U.S. Provisional Application No. 61/648,041,entitled, “COMPOSITIONS AND METHODS FOR MODULATING PTEN EXPRESSION”,filed on May 16, 2012, of U.S. Provisional Application No. 61/785,885,entitled, “COMPOSITIONS AND METHODS FOR MODULATING PTEN EXPRESSION”,filed on Mar. 14, 2013, of U.S. Provisional Application No. 61/648,058,entitled, “COMPOSITIONS AND METHODS FOR MODULATING BDNF EXPRESSION”,filed on May 16, 2012, and of U.S. Provisional Application No.61/648,051, entitled, “COMPOSITIONS AND METHODS FOR MODULATING MECP2EXPRESSION”, filed on May 16, 2012, the contents of each of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to oligonucleotide based compositions, as well asmethods of using oligonucleotide based compositions for treatingdisease.

BACKGROUND OF THE INVENTION

Spinal muscular atrophy (SMA) is a group of hereditary diseases thatcauses muscle damage leading to impaired muscle function, difficultybreathing, frequent respiratory infection, and eventually death. Thereare four types of SMA that are classified based on the onset andseverity of the disease. SMA type I is the most severe form and is oneof the most common causes of infant mortality, with symptoms of muscleweakness and difficulty breathing occurring at birth. SMA type II occurslater, with muscle weakness and other symptoms developing from ages 6month to 2 years. Symptoms appear in SMA type III during childhood andin SMA type IV, the mildest form, during adulthood. All four types ofSMA have been found to be associated with mutations in the SMN genefamily, particularly SMN1.

Survival of motor neuron (SMN) is a protein involved in transcriptionalsplicing through its involvement in assembly of small nuclearribonucleoproteins (snRNPs). snRNPs are protein-RNA complexes that bindwith pre-mRNA to form a spliceosome, which then splices the pre-mRNA,most often resulting in removal of introns. Three genes encode SMN or avariant of SMN, including SMN1 (survival of motor neuron 1, telomeric),SMN2 (survival of motor neuron 2, centromeric), and SMNP (survival ofmotor neuron 1, telomeric pseudogene). SMN1 and SMN2 are a result of agene duplication at 5q13 in humans. A lack of SMN activity results inwidespread splicing defects, especially in spinal motor neurons, anddegeneration of the spinal cord lower motor neurons.

SUMMARY OF THE INVENTION

Aspects of the invention disclosed herein provide methods andcompositions that are useful for upregulating the expression of certaingenes in cells. In some embodiments, single stranded oligonucleotidesare provided that target a PRC2-associated region of a gene and therebycause upregulation of the gene. Also provided herein are methods andrelated single stranded oligonucleotides that are useful for selectivelyinducing expression of particular splice variants of genes. In someembodiments, the methods are useful for controlling the levels in a cellof particular protein isoforms encoded by the splice variants. In somecases, the methods are useful for inducing expression of proteins tolevels sufficient to treat disease.

In some embodiments, single stranded oligonucleotides are provided thattarget a PRC2-associated region of a SMN gene (e.g., human SMN1, humanSMN2) and thereby cause upregulation of the gene. For example, accordingto some aspects of the invention methods are provided for increasingexpression of full-length SMN protein in a cell for purposes of treatingSMA. Accordingly, aspects of the invention disclosed herein providemethods and compositions that are useful for upregulating SMN1 or SMN2in cells. In some embodiments, single stranded oligonucleotides areprovided that target a PRC2-associated region of the gene encoding SMN1or SMN2. In some embodiments, these single stranded oligonucleotidesactivate or enhance expression of SMN1 or SMN2 by relieving orpreventing PRC2 mediated repression of SMN1 or SMN2.

In some embodiments, the methods comprise delivering to the cell a firstsingle stranded oligonucleotide complementary with a PRC2-associatedregion of an SMN gene, e.g., a PRC2-associated region of SMN1 or SMN2,and a second single stranded oligonucleotide complementary with a splicecontrol sequence of a precursor mRNA of an SMN gene, e.g., a precursormRNA of SMN1 or SMN2, in amounts sufficient to increase expression of amature mRNA of SMN1 or SMN2 that comprises (or includes) exon 7 in thecell.

According to some aspects of the invention single strandedoligonucleotides are provided that have a region of complementarity thatis complementarity with (e.g., at least 8 consecutive nucleotides of) aPRC2-associated region of an SMN gene, e.g., a PRC2-associated region ofthe nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5. In someembodiments, the oligonucleotide has at least one of the followingfeatures: a) a sequence that is 5′X-Y-Z, in which X is any nucleotideand in which X is at the 5′ end of the oligonucleotide, Y is anucleotide sequence of 6 nucleotides in length that is not a human seedsequence of a microRNA, and Z is a nucleotide sequence of 1 to 23nucleotides in length; b) a sequence that does not comprise three ormore consecutive guanosine nucleotides; c) a sequence that has less thana threshold level of sequence identity with every sequence ofnucleotides, of equivalent length to the second nucleotide sequence,that are between 50 kilobases upstream of a 5′-end of an off-target geneand 50 kilobases downstream of a 3′-end of the off-target gene; d) asequence that is complementary to a PRC2-associated region that encodesan RNA that forms a secondary structure comprising at least two singlestranded loops; and e) a sequence that has greater than 60% G-C content.In some embodiments, the single stranded oligonucleotide has at leasttwo of features a), b), c), d), and e), each independently selected. Insome embodiments, the single stranded oligonucleotide has at least threeof features a), b), c), d), and e), each independently selected. In someembodiments, the single stranded oligonucleotide has at least four offeatures a), b), c), d), and e), each independently selected. In someembodiments, the single stranded oligonucleotide has each of featuresa), b), c), d), and e). In certain embodiments, the oligonucleotide hasthe sequence 5′X-Y-Z, in which the oligonucleotide is 8-50 nucleotidesin length.

According to some aspects of the invention, single strandedoligonucleotides are provided that have a sequence X-Y-Z, in which X isany nucleotide, Y is a nucleotide sequence of 6 nucleotides in lengththat is not a seed sequence of a human microRNA, and Z is a nucleotidesequence of 1 to 23 nucleotides in length, in which the single strandedoligonucleotide is complementary with a PRC2-associated region of an SMNgene, e.g., a PRC2-associated region of the nucleotide sequence setforth as SEQ ID NO: 1, 2, 4, or 5. In some aspects of the invention,single stranded oligonucleotides are provided that have a sequence5′-X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequence of 6nucleotides in length that is not a seed sequence of a human microRNA,and Z is a nucleotide sequence of 1 to 23 nucleotides in length, inwhich the single stranded oligonucleotide is complementary with at least8 consecutive nucleotides of a PRC2-associated region of an SMN gene,e.g., a PRC2-associated region of the nucleotide sequence set forth asSEQ ID NO: 1, 2, 4, or 5. In some embodiments, Y is a sequence selectedfrom Table 1. In some embodiments, the PRC2-associated region is asequence listed in any one of SEQ ID NOS: 9 to 18.

In some embodiments, the single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087,or a fragment thereof that is at least 8 nucleotides. In someembodiments, the single stranded oligonucleotide comprises a nucleotidesequence as set forth in any one of SEQ ID NOS: 30 to 13087, in whichthe 5′ end of the nucleotide sequence provided is the 5′ end of theoligonucleotide. In some embodiments, the region of complementarity(e.g., the at least 8 consecutive nucleotides) is also present withinthe nucleotide sequence set forth as SEQ ID NO: 7 or 8.

In some embodiments, a PRC2-associated region is a sequence listed inany one of SEQ ID NOS: 9 to 14. In some embodiments, the single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof thatis at least 8 nucleotides. In some embodiments, the single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5′ end of thenucleotide sequence provided is the 5′ end of the oligonucleotide. Insome embodiments, the at least 8 consecutive nucleotides are alsopresent within the nucleotide sequence set forth as SEQ ID NO: 7.

In some embodiments, a PRC2-associated region is a sequence listed inany one of SEQ ID NOS: 15 to 18. In some embodiments, the singlestranded oligonucleotide comprises a nucleotide sequence as set forth inany one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471,2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918,4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 ora fragment thereof that is at least 8 nucleotides. In some embodiments,the at least 8 consecutive nucleotides are present within the nucleotidesequence set forth as SEQ ID NO: 8.

In some embodiments, the single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 30 to 13087.In some embodiments, the oligonucleotide is up to 50 nucleotides inlength. In some embodiments, the single stranded oligonucleotidecomprises a fragment of at least 8 nucleotides of a nucleotide sequenceas set forth in any one of SEQ ID NOS: 30 to 13087.

In some embodiments, a single stranded oligonucleotide comprises anucleotide sequence as set forth in Table 4. In some embodiments, thesingle stranded oligonucleotide comprises a fragment of at least 8nucleotides of a nucleotide sequence as set forth in Table 4. In someembodiments, a single stranded oligonucleotide consists of a nucleotidesequence as set forth in Table 4.

According to some aspects of the invention, compounds are provided thatcomprise the general formula A-B-C, wherein A is a single strandedoligonucleotide complementary with at least 8 consecutive nucleotides ofa PRC2-associated region of a gene, B is a linker, and C is a singlestranded oligonucleotide complementary to a splice control sequence of aprecursor mRNA of the gene. In some embodiments, B comprises anoligonucleotide, peptide, low pH labile bond, or disulfide bond. In someembodiments, the splice control sequence resides in an exon of the gene.In some embodiments, the splice control sequence traverses anintron-exon junction of the gene. In some embodiments, the splicecontrol sequence resides in an intron of the gene. In some embodiments,the splice control sequence comprises at least one hnRNAP bindingsequence. In some embodiments, hybridization of an oligonucleotidehaving the sequence of C with the splice control sequence of theprecursor mRNA in a cell results in inclusion of a particular exon in amature mRNA that arises from processing of the precursor mRNA in thecell. In some embodiments, hybridization of an oligonucleotide havingthe sequence of C with the splice control sequence of the precursor mRNAin a cell results in exclusion of a particular intron or exon in amature mRNA that arises from processing of the precursor mRNA in thecell.

In some embodiments, the gene is SMN1 or SMN2. In some embodiments, thesplice control sequence resides in intron 6, intron 7, exon 7, exon 8 orat the junction of intron 7 and exon 8 of SMN1 or SMN2. In someembodiments, the splice control sequence comprises the sequence:CAGCAUUAUGAAAG (SEQ ID NO: 13100). In some embodiments, B comprises asequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088);TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090);ACTTTCATAATGCTG (SEQ ID NO: 13090); and CTTTCATAATGCTGG (SEQ ID NO:13092).

In some embodiments, A has a sequence 5′-X-Y-Z, wherein X is anynucleotide, Y is a nucleotide sequence of 6 nucleotides in length thatis not a seed sequence of a human microRNA, and Z is a nucleotidesequence of 1-23 nucleotides in length. In some embodiments, thePRC2-associated region of an SMN2 gene is a PRC2-associated regionwithin SEQ ID NO: 1, 2, 4 or 5. In some embodiments, Y is a sequenceselected from Table 1. In some embodiments, the PRC2-associated regionis a sequence set forth in any one of SEQ ID NOS: 9 to 23. In someembodiments, A comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30 to 8329 and 13088 to 13094 or a fragment thereof thatis at least 8 nucleotides. In some embodiments, A comprises a nucleotidesequence as set forth in any one of SEQ ID NOS: 30 to 8329 and 13088 to13094, wherein the 5′ end of the nucleotide sequence provided is the 5′end of A. In some embodiments, the at least 8 consecutive nucleotidesare also present within the nucleotide sequence set forth as SEQ ID NO:7. In some embodiments, the PRC2-associated region is a sequence setforth in any one of SEQ ID NOS: 24 to 29.

In some embodiments, A comprises a nucleotide sequence as set forth inany one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471,2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918,4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and 13062-13087 ora fragment thereof that is at least 8 nucleotides. In some embodiments,the at least 8 consecutive nucleotides are present within the nucleotidesequence set forth as SEQ ID NO: 8. In some embodiments, A does notcomprise three or more consecutive guanosine nucleotides. In someembodiments, A does not comprise four or more consecutive guanosinenucleotides. In some embodiments, A or C is 8 to 30 nucleotides inlength. In some embodiments, A is 8 to 10 nucleotides in length and allbut 1, 2, or 3 of the nucleotides of the complementary sequence of thePRC2-associated region are cytosine or guanosine nucleotides. In someembodiments, B is an oligonucleotide comprising 1 to 10 thymidines oruridines. In some embodiments, B is more susceptible to cleavage in amammalian extract than A and C.

In some embodiments, A comprises a nucleotide sequence selected fromGCTUTGGGAAGUAUG (SEQ ID NO: 11394), CUTUGGGAAGTATG (SEQ ID NO: 11395)and GGTACATGAGTGGCT (SEQ ID NO: 11419); B comprises the nucleotidesequence TTTT or UUUU; and C comprises the nucleotide sequenceTCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO:13089); CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO:13091): or CTTTCATAATGCTGG (SEQ ID NO: 13092), and wherein the 3′ end ofA is linked to the 5′ end of B, and the 3′ end of B is linked to 5′ endof C.

In some embodiments, the single stranded oligonucleotide does notcomprise three or more consecutive guanosine nucleotides. In someembodiments, the single stranded oligonucleotide does not comprise fouror more consecutive guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide is 8 to 30nucleotides in length. In some embodiments, the single strandedoligonucleotide is up to 50 nucleotides in length. In some embodiments,the single stranded oligonucleotide is 8 to 10 nucleotides in length andall but 1, 2, or 3 of the nucleotides of the complementary sequence ofthe PRC2-associated region are cytosine or guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide iscomplementary with at least 8 consecutive nucleotides of aPRC2-associated region of an SMN gene, e.g., a PRC2-associated region ofa nucleotide sequence set forth as SEQ ID NO: 1, 2, 4, or 5, in whichthe nucleotide sequence of the single stranded oligonucleotide comprisesone or more of a nucleotide sequence selected from the group consistingof

(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,

(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx,(X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX,(X)xxxXXx, (X)xxxXxX and (X)xxxxXX,

(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx,(X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,

(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx, (X)XxxXXXX,(X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx,(X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,

(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx,and

(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx,wherein “X” denotes a nucleotide analogue, (X) denotes an optionalnucleotide analogue, and “x” denotes a DNA or RNA nucleotide unit.

In some embodiments, at least one nucleotide of the oligonucleotide is anucleotide analogue. In some embodiments, the at least one nucleotideanalogue results in an increase in Tm of the oligonucleotide in a rangeof 1 to 5° C. compared with an oligonucleotide that does not have the atleast one nucleotide analogue.

In some embodiments, at least one nucleotide of the oligonucleotidecomprises a 2′ O-methyl. In some embodiments, each nucleotide of theoligonucleotide comprises a 2′ O-methyl. In some embodiments, theoligonucleotide comprises at least one ribonucleotide, at least onedeoxyribonucleotide, or at least one bridged nucleotide. In someembodiments, the bridged nucleotide is a LNA nucleotide, a cEtnucleotide or a ENA modified nucleotide. In some embodiments, eachnucleotide of the oligonucleotide is a LNA nucleotide.

In some embodiments, the nucleotides of the oligonucleotide comprisealternating deoxyribonucleotides and 2′-fluoro-deoxyribonucleotides. Insome embodiments, the nucleotides of the oligonucleotide comprisealternating deoxyribonucleotides and 2′-O-methyl nucleotides. In someembodiments, the nucleotides of the oligonucleotide comprise alternatingdeoxyribonucleotides and ENA nucleotide analogues. In some embodiments,the nucleotides of the oligonucleotide comprise alternatingdeoxyribonucleotides and LNA nucleotides. In some embodiments, the 5′nucleotide of the oligonucleotide is a deoxyribonucleotide. In someembodiments, the nucleotides of the oligonucleotide comprise alternatingLNA nucleotides and 2′-O-methyl nucleotides. In some embodiments, the 5′nucleotide of the oligonucleotide is a LNA nucleotide. In someembodiments, the nucleotides of the oligonucleotide comprisedeoxyribonucleotides flanked by at least one LNA nucleotide on each ofthe 5′ and 3′ ends of the deoxyribonucleotides.

In some embodiments, the single stranded oligonucleotide comprisesmodified internucleotide linkages (e.g., phosphorothioateinternucleotide linkages or other linkages) between at least two, atleast three, at least four, at least five or more nucleotides. In someembodiments, the single stranded oligonucleotide comprises modifiedinternucleotide linkages (e.g., phosphorothioate internucleotidelinkages or other linkages) between all nucleotides.

In some embodiments, the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group. In some embodiments, thenucleotide at the 3′ position of the oligonucleotide has a 3′thiophosphate. In some embodiments, the single stranded oligonucleotidehas a biotin moiety or other moiety conjugated to its 5′ or 3′nucleotide. In some embodiments, the single stranded oligonucleotide hascholesterol, Vitamin A, folate, sigma receptor ligands, aptamers,peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR ordynamic polyconjugates and variants thereof at its 5′ or 3′ end.

According to some aspects of the invention compositions are providedthat comprise any of the oligonucleotides disclosed herein, and acarrier. In some embodiments, compositions are provided that compriseany of the oligonucleotides in a buffered solution. In some embodiments,the oligonucleotide is conjugated to the carrier. In some embodiments,the carrier is a peptide. In some embodiments, the carrier is a steroid.According to some aspects of the invention pharmaceutical compositionsare provided that comprise any of the oligonucleotides disclosed herein,and a pharmaceutically acceptable carrier.

According to other aspects of the invention, kits are provided thatcomprise a container housing any of the compositions disclosed herein.

According to some aspects of the invention, methods of increasingexpression of SMN1 or SMN2 in a cell are provided. In some embodiments,the methods involve delivering any one or more of the single strandedoligonucleotides disclosed herein into the cell. In some embodiments,delivery of the single stranded oligonucleotide into the cell results ina level of expression of SMN1 or SMN2 that is greater (e.g., at least50% greater) than a level of expression of SMN1 or SMN2 in a controlcell that does not comprise the single stranded oligonucleotide.

According to some aspects of the invention, methods of increasing levelsof SMN1 or SMN2 in a subject are provided. According to some aspects ofthe invention, methods of treating a condition (e.g., Spinal muscularatrophy) associated with decreased levels of SMN1 or SMN2 in a subjectare provided. In some embodiments, the methods involve administering anyone or more of the single stranded oligonucleotides disclosed herein tothe subject.

Aspects of the invention relate to methods of increasing expression ofSMN protein in a cell. In some embodiments, the method comprisedelivering to the cell a first single stranded oligonucleotidecomplementary with at least 8 consecutive nucleotides of aPRC2-associated region of SMN2 and a second single strandedoligonucleotide complementary with a splice control sequence of aprecursor mRNA of SMN2, in amounts sufficient to increase expression ofa mature mRNA of SMN2 that comprises exon 7 in the cell. In someembodiments, the region of complementarity with at least 8 consecutivenucleotides of a PRC2-associated region of SMN2 has at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least8, or more mismatches with a corresponding region of SMN1. As usedherein the term, “splice control sequence” refers to a nucleotidesequence that when present in a precursor mRNA influences splicing ofthat precursor mRNA in a cell. In some embodiments, a splice controlsequence includes one or more binding sites for a molecule thatregulates mRNA splicing, such as a hnRNAP protein. In some embodiments,a splice control sequence comprises the sequence CAG or AAAG. In someembodiments, a splice control sequence resides in an exon (e.g., an exonof SMN1 or SMN2, such as exon 7 or exon 8). In some embodiments, asplice control sequence traverses an intron-exon junction (e.g., anintron-exon junction of SMN1 or SMN2, such as the intron 6/exon 7junction or the intron 7/exon 8 junction). In some embodiments, a splicecontrol sequence resides in an intron (e.g., an intron of SMN1 or SMN2,such as intron 6 or intron 7). In some embodiments, a splice controlsequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100) or aportion thereof.

In some embodiments, the second single stranded oligonucleotide issplice switching oligonucleotide that comprises a sequence selectedfrom: TCACTTTCATAATGCTGG (SEQ ID NO: 13088); TCACTTTCATAATGC (SEQ ID NO:13089); CACTTTCATAATGCT (SEQ ID NO: 13090); ACTTTCATAATGCTG (SEQ ID NO:13091); and CTTTCATAATGCTGG (SEQ ID NO: 13092). In some embodiments, thesecond single stranded oligonucleotide is 8 to 30 nucleotides in length.

In some embodiments, the first single stranded oligonucleotide has asequence 5′-X-Y-Z, wherein X is any nucleotide, Y is a nucleotidesequence of 6 nucleotides in length that is not a seed sequence of ahuman microRNA, and Z is a nucleotide sequence of 1-23 nucleotides inlength. In some embodiments, the PRC2-associated region of an SMN2 geneis a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5. In someembodiments, Y is a sequence selected from Table 1. In some embodiments,the PRC2-associated region is a sequence set forth in any one of SEQ IDNOS: 9 to 23. In some embodiments, the first single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30 to 8329 and 13088 to 13094 or a fragment thereof thatis at least 8 nucleotides. In some embodiments, the first singlestranded oligonucleotide comprises a nucleotide sequence as set forth inany one of SEQ ID NOS: 30 to 8329 and 13088 to 13094, wherein the 5′ endof the nucleotide sequence provided is the 5′ end of the first singlestranded oligonucleotide. In some embodiments, the at least 8consecutive nucleotides are also present within the nucleotide sequenceset forth as SEQ ID NO: 7.

In some embodiments, the PRC2-associated region is a sequence set forthin any one of SEQ ID NOS: 24 to 29. In some embodiments, the firstsingle stranded oligonucleotide comprises a nucleotide sequence as setforth in any one of SEQ ID NOS: 1158-1159, 1171, 1482-1483, 1485-1486,2465-2471, 2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440,3916-3918, 4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, and13062-13087 or a fragment thereof that is at least 8 nucleotides. Insome embodiments, the at least 8 consecutive nucleotides are presentwithin the nucleotide sequence set forth as SEQ ID NO: 8. In someembodiments, the first single stranded oligonucleotide does not comprisethree or more consecutive guanosine nucleotides. In some embodiments,the first single stranded oligonucleotide does not comprise four or moreconsecutive guanosine nucleotides. In some embodiments, the first singlestranded oligonucleotide is 8 to 30 nucleotides in length. In someembodiments, the first single stranded oligonucleotide is 8 to 10nucleotides in length and all but 1, 2, or 3 of the nucleotides of thecomplementary sequence of the PRC2-associated region are cytosine orguanosine nucleotides.

In some embodiments, the first single stranded oligonucleotide and thesecond single stranded oligonucleotide are delivered to the cellsimultaneously. In some embodiments, the cell is in a subject and thestep of delivering to the cell comprises administering the first singlestranded oligonucleotide and the second single stranded oligonucleotideto the subject as a co-formulation. In some embodiments, the firstsingle stranded oligonucleotide is covalently linked to the secondsingle stranded oligonucleotide through a linker. In some embodiments,the linker comprises an oligonucleotide, a peptide, a low pH-labilebond, or a disulfide bond. In some embodiments, the linker comprises anoligonucleotide, optionally wherein the oligonucleotide comprises 1 to10 thymidines or uridines. In some embodiments, the linker is moresusceptible to cleavage in a mammalian extract than the first and secondsingle stranded oligonucleotides. In some embodiments, the first singlestranded oligonucleotide is not covalently linked to the second singlestranded oligonucleotide. In some embodiments, the first single strandedoligonucleotide and the second single stranded oligonucleotide aredelivered to the cell separately.

According to some aspects of the invention, methods are provided fortreating spinal muscular atrophy in a subject. The methods, in someembodiments, comprise administering to the subject a first singlestranded oligonucleotide complementary with at least 8 consecutivenucleotides of a PRC2-associated region of SMN2 and a second singlestranded oligonucleotide complementary with a splice control sequence ofa precursor mRNA of SMN2, in amounts sufficient to increase expressionof SMN protein in the subject.

According to some aspects of the invention methods are provided fortreating spinal muscular atrophy in a subject that involve administeringto the subject a first single stranded oligonucleotide complementarywith a PRC2-associated region of SMN2 and a second single strandedoligonucleotide complementary with a splice control sequence of aprecursor mRNA of SMN2, in amounts sufficient to increase expression ofSMN protein in the subject. Related compositions are also provided. Insome embodiments, compositions are provided that comprise a first singlestranded oligonucleotide complementary with at least 8 consecutivenucleotides of a PRC2-associated region of SMN2, and a second singlestranded oligonucleotide complementary to a splice control sequence of aprecursor mRNA of SMN2. In some embodiments, compositions are providedthat comprise a single stranded oligonucleotide complementary with atleast 8 consecutive nucleotides of a PRC2-associated region of a gene,linked via a linker to a single stranded oligonucleotide complementaryto a splice control sequence of a precursor mRNA of the gene. Relatedkits comprising single stranded oligonucleotides that regulate SMN1 orSMN2 expression are also provided.

According to some aspects of the invention compositions are providedthat comprise any of the oligonucleotides or compounds disclosed herein,and a carrier. In some embodiments, compositions are provided thatcomprise any of the oligonucleotides or compounds in a bufferedsolution. In some embodiments, the oligonucleotide is conjugated to thecarrier. In some embodiments, the carrier is a peptide. In someembodiments, the carrier is a steroid. According to some aspects of theinvention pharmaceutical compositions are provided that comprise any ofthe oligonucleotides disclosed herein, and a pharmaceutically acceptablecarrier.

According to some aspects of the invention, compositions are providedthat comprise a first single stranded oligonucleotide complementary withat least 8 consecutive nucleotides of a PRC2-associated region of SMN2,and a second single stranded oligonucleotide complementary to a splicecontrol sequence of a precursor mRNA of SMN2. In some embodiments, thesplice control sequence resides in an exon of SMN2. In some embodiments,the exon is exon 7 or exon 8. In some embodiments, the splice controlsequence traverses an intron-exon junction of SMN2. In some embodiments,the intron-exon junction is the intron 6/exon 7 junction or the intron7/exon 8 junction. In some embodiments, the splice control sequenceresides in an intron of SMN2. In some embodiments, the intron is intron6 or intron 7 (SEQ ID NO: 13101). In some embodiments, the splicecontrol sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO:13100) or a portion thereof. In some embodiments, the splice controlsequence comprises at least one hnRNAP binding sequence. In someembodiments, the second single stranded oligonucleotide comprises asequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088);TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090);ACTTTCATAATGCTG (SEQ ID NO: 13091); and CTTTCATAATGCTGG (SEQ ID NO:13092). In some embodiments, the first single stranded oligonucleotidehas a sequence 5′-X-Y-Z, wherein X is any nucleotide, Y is a nucleotidesequence of 6 nucleotides in length that is not a seed sequence of ahuman microRNA, and Z is a nucleotide sequence of 1-23 nucleotides inlength. In some embodiments, the PRC2-associated region of SMN2 is aPRC2-associated region within SEQ ID NO: 1, 2, 4 or 5. In someembodiments, Y is a sequence selected from Table 1. In some embodiments,the PRC2-associated region is a sequence set forth in any one of SEQ IDNOS: 9 to 23. In some embodiments, the first single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof thatis at least 8 nucleotides. In some embodiments, the first singlestranded oligonucleotide comprises a nucleotide sequence as set forth inany one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5′ endof the nucleotide sequence provided is the 5′ end of the first singlestranded oligonucleotide. In some embodiments, the at least 8consecutive nucleotides are also present within the nucleotide sequenceset forth as SEQ ID NO: 7. In some embodiments, the PRC2-associatedregion is a sequence set forth in any one of SEQ ID NOS: 24 to 29. Insome embodiments, the first single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159,1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657,2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061,7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least8 nucleotides. In some embodiments, the at least 8 consecutivenucleotides are present within the nucleotide sequence set forth as SEQID NO: 8. In some embodiments, the first single stranded oligonucleotidedoes not comprise three or more consecutive guanosine nucleotides. Insome embodiments, the first single stranded oligonucleotide does notcomprise four or more consecutive guanosine nucleotides. In someembodiments, the first and/or second single stranded oligonucleotide is8 to 30 nucleotides in length. In some embodiments, the first singlestranded oligonucleotide is 8 to 10 nucleotides in length and all but 1,2, or 3 of the nucleotides of the complementary sequence of thePRC2-associated region are cytosine or guanosine nucleotides. In someembodiments, the first single stranded oligonucleotide is covalentlylinked to the second single stranded oligonucleotide through a linker.In some embodiments, the linker comprises an oligonucleotide, a peptide,a low pH-labile bond, or a disulfide bond. In some embodiments, thelinker comprises an oligonucleotide, optionally wherein theoligonucleotide comprises 1 to 10 thymidines or uridines. In someembodiments, the linker is more susceptible to cleavage in a mammalianextract than the first and second single stranded oligonucleotides. Insome embodiments, the first single stranded oligonucleotide is notcovalently linked to the second single stranded oligonucleotide. In someembodiments, the composition further comprises a carrier. In someembodiments, the carrier is a pharmaceutically acceptable carrier.

Further aspects of the invention provide methods for selectingoligonucleotides for activating or enhancing expression of SMN1 or SMN2.In some embodiments, methods are provided for selecting a set ofoligonucleotides that is enriched in candidates (e.g., compared with arandom selection of oligonucleotides) for activating or enhancingexpression of SMN1 or SMN2. Accordingly, the methods may be used toestablish sets of clinical candidates that are enriched inoligonucleotides that activate or enhance expression of SMN1 or SMN2.Such libraries may be utilized, for example, to identify leadoligonucleotides for developing therapeutics to treat SMN1 or SMN2.Furthermore, in some embodiments, oligonucleotide chemistries areprovided that are useful for controlling the pharmacokinetics,biodistribution, bioavailability and/or efficacy of the single strandedoligonucleotides for activating expression of SMN1 or SMN2.

According to other aspects of the invention, kits are provided thatcomprise a container housing any of the compositions disclosed herein.According to other aspects of the invention, kits are provided thatcomprise a first container housing first single stranded oligonucleotidecomplementary with at least 8 consecutive nucleotides of aPRC2-associated region of a gene; and a second container housing asecond single stranded oligonucleotide complementary to a splice controlsequence of a precursor mRNA of the gene. In some embodiments, thesplice control sequence resides in an exon of the gene. In someembodiments, the splice control sequence traverses an intron-exonjunction of the gene. In some embodiments, the splice control sequenceresides in an intron of the gene. In some embodiments, the splicecontrol sequence comprises at least one hnRNAP binding sequence. In someembodiments, hybridization of an oligonucleotide having the sequence ofC with the splice control sequence of the precursor mRNA in a cellresults in inclusion of a particular exon in a mature mRNA that arisesfrom processing of the precursor mRNA in the cell. In some embodiments,hybridization of an oligonucleotide having the sequence of C with thesplice control sequence of the precursor mRNA in a cell results inexclusion of a particular intron or exon in a mature mRNA that arisesfrom processing of the precursor mRNA in the cell. In some embodiments,the gene is SMN1 or SMN2. In some embodiments, the splice controlsequence resides in intron 6, intron 7, exon 7, exon 8 or at thejunction of intron 7 and exon 8. In some embodiments, the splice controlsequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO: 13100). Insome embodiments, the second single stranded oligonucleotide comprises asequence selected from: TCACTTTCATAATGCTGG (SEQ ID NO: 13088);TCACTTTCATAATGC (SEQ ID NO: 13089); CACTTTCATAATGCT (SEQ ID NO: 13090);ACTTTCATAATGCTG (SEQ ID NO: XX); and CTTTCATAATGCTGG (SEQ ID NO: 13091).In some embodiments, the first single stranded oligonucleotide has asequence 5′-X-Y-Z, wherein X is any nucleotide, Y is a nucleotidesequence of 6 nucleotides in length that is not a seed sequence of ahuman microRNA, and Z is a nucleotide sequence of 1-23 nucleotides inlength. In some embodiments, the PRC2-associated region of an SMN2 geneis a PRC2-associated region within SEQ ID NO: 1, 2, 4 or 5. In someembodiments, Y is a sequence selected from Table 1. In some embodiments,the PRC2-associated region is a sequence set forth in any one of SEQ IDNOS: 9 to 23. In some embodiments, the first single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30 to 8329 and 13093 to 13094 or a fragment thereof thatis at least 8 nucleotides. In some embodiments, the first singlestranded oligonucleotide comprises a nucleotide sequence as set forth inany one of SEQ ID NOS: 30 to 8329 and 13093 to 13094, wherein the 5′ endof the nucleotide sequence provided is the 5′ end of the first singlestranded oligonucleotide. In some embodiments, the at least 8consecutive nucleotides are also present within the nucleotide sequenceset forth as SEQ ID NO: 7. In some embodiments, the PRC2-associatedregion is a sequence set forth in any one of SEQ ID NOS: 24 to 29. Insome embodiments, the first single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 1158-1159,1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657,2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061,7327, 8330-13061, and 13062-13087 or a fragment thereof that is at least8 nucleotides. In some embodiments, the at least 8 consecutivenucleotides are present within the nucleotide sequence set forth as SEQID NO: 8.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a schematic of SMN1 and SMN2 mRNA processing

FIG. 2 provides a table outlining genotypes and patent information,including SMA classification, of cell lines tested in Example 2.Baseline SMN protein levels in the cell lines are also depicted.

FIG. 3 depicts results of RT-PCR assays showing effects on SMN mRNAexpression of oligonucleotides directed against a PRC2-associated regionof SMN2 (oligos 1-52 and 59-101) and splice switching oligonucleotides(oligos 53-58) (PCR primers directed against exon 1 of SMN1/2.) in cellline 3814.

FIG. 4 depicts results of RT-PCR assays showing effects on SMN mRNAexpression of oligonucleotides directed against a PRC2-associated regionof SMN2 (oligos 1-52 and 59-101) and splice switching oligonucleotides(oligos 53-58) (PCR primers directed against exon 1 of SMN1/2.) in cellline 3813.

FIG. 5 shows that splice switching oligonucleotides (oligos 53-58)increase expression of full length SMN2. Results are based on a gelseparation analysis of PCR products obtained following a DdeIrestriction digest. Two cell lines were tested, 3813 and 9677. Oligo 84,which targets a PRC2-associated region of SMN2, did not exhibit anincrease in full length SMN2 expression when delivered alone to cells.

FIGS. 6A-6B provide the results of an SMN ELISA (Enzo) showing thatcertain oligonucleotides directed against a PRC2-associated region ofSMN2 alone do not significantly increase SMN2 protein 24 hpost-transfection in certain SMA patient fibroblasts (compared toLipofectamine treated cells—dashed line).

FIGS. 7A-7B provide the results of an SMN ELISA showing thatoligonucleotides directed against a PRC2-associated region of SMN2 incombination with a splice switching oligonucleotide (oligo 53)significantly increase SMN2 protein 24 h post-transfection in SMApatient fibroblasts (compared to Lipofectamine treated cells—dashedline).

FIGS. 8A-8B provide the results of an SMN ELISA showing thatoligonucleotides directed against a PRC2-associated region of SMN2 incombination with a splice switching oligonucleotide (oligo 54)significantly increase SMN2 protein 24 h post-transfection in SMApatient fibroblasts (compared to Lipofectamine treated cells—dashedline).

FIG. 9 provides results of an RT-PCR assay showing that oligonucleotidesdirected against a PRC2-associated region of SMN2 in combination with asplice switching oligonucleotide (oligo 53 or 54) significantly increaseSMN2 protein 24 h post-transfection in SMA patient fibroblasts (comparedto negative control oligo and Lipofectamine treated cells). LNA/2′OMealternating oligonucleotide (LM design) and DNA/LNA alternatingoligonucleotides (DL design) were tested.

BRIEF DESCRIPTION OF TABLES

Table 1: Hexamers that are not seed sequences of human miRNAs

Table 2: Oligonucleotide sequences made for testing in the lab. RQ(column 2) and RQ SE (column 3) shows the activity of the oligo relativeto a control well (usually carrier alone) and the standard error or thetriplicate replicates of the experiment. [oligo] is shown in nanomolarfor in vitro experiments and in milligrams per kilogram of body weightfor in vivo experiments.

Table 3: A listing of oligonucleotide modifications

Table 4: Oligonucleotide sequences made for testing human cells obtainedfrom subjects with Spinal Muscular Atrophy. The table shows the sequenceof the modified nucleotides, where lnaX represents an LNA nucleotidewith 3′ phosphorothioate linkage, omeX is a 2′-O-methyl nucleotide, dXis a deoxy nucleotide. An s at the end of a nucleotide code indicatesthat the nucleotide had a 3′ phosphorothioate linkage. The “-Sup” at theend of the sequence marks the fact that the 3′ end lacks either aphosphate or thiophosphate on the 3′ linkage. The Formatted Sequencecolumn shows the sequence of the oligonucleotide, including modifiednucleotides, for the oligonucleotides tested in Table 2, 5, 6 and 7.

Table 8: Cell lines

BRIEF DESCRIPTION OF THE APPENDICES

-   -   Appendix A; Appendix A contains Table 5, which shows RT-PCR data        from PGPubs, use the testing of different oligonucleotides.        Appendix A can be found on pages 106-287 Gap Bulletin per of WO        2013/173638, which are incorporated by reference herein in their        entirety.    -   Appendix B; Appendix B contains Table 6, which shows RT-PCR data        from testing of different combination treatments (e.g., two        oligonucleotides, an oligonucleotide and a drug). Appendix B can        be found on pages 288-309 of WO 2013/173638, which are        incorporated by reference herein in their entirety.    -   Appendix C; Appendix C contains Table 7, which shows ELISA data        from testing of different oligonucleotides. Appendix C can be        found on pages 310-399 of WO 2013/173638, which are incorporated        by reference herein in their entirety.

Note the following column information for Tables 5-7 in Appendices A-C,respectively. SEQID: sequence identifier of base sequence ofoligonucleotide used; Oligo Name: name of oligonucleotide; Avg RQ:average relative quantification of RT-PCR based expression levels oftarget gene(s); Avg RQ SE: standard error of mean of relativequantification of RT-PCR based expression level; “% SMN over lipo onlycontrol” refers to the ratio of SMN protein levels (ng/mg total protein)when compared to Lipofectamine2000 (transfection reagent) treated cellsconverted into %; “% SMN CVV” refers to coefficient of variation; Exp #:Experiment reference number; Target: target gene; [oligo]: concentrationof oligonucleotide used in nM unless otherwise indicated; Cell Line:cell line used; Assay Type: assay used; Time(hr): time of assayfollowing treatment; 2^(nd) Drug: name of second oligonucleotide(identified by Oligo Name) or drug used in combination experiment;[2^(nd)]: concentration of second oligonucleotide or drug; Units: unitsof concentration; 3^(rd) Drug: name of third oligonucleotide (identifiedby Oligo Name) or drug used in combination experiment; [3^(rd)]:concentration of third oligonucleotide or drug; Notes: commentsregarding experiment. Oligo Names correspond to those in Tables 2 and 4.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Spinal muscular atrophy (SMA), the most common genetic cause of infantmortality, is an autosomal recessive neuromuscular disease characterizedby progressive loss of α-motor neurons in the anterior horns of thespinal cord, leading to limb and trunk paralysis and atrophy ofvoluntary muscles. Based on the severity and age of onset, SMA isclinically subdivided into types I, II, and III (MIMs 253300, 253550,and 253400), with type I generally understood as being the most severe.

Loss of function of the SMN1 gene is responsible for SMA. Humans have anextra SMN gene copy, called SMN2. Both SMN genes reside within asegmental duplication on Chromosome 5q13 as inverted repeats. SMN1 andSMN2 are almost identical. In some cases, SMN1 and SMN2 differ by 11nucleotide substitutions, including seven in intron 6, two in intron 7,one in coding exon 7, and one in non-coding exon 8. The substitution inexon 7 involves a translationally silent C to T transition compared withSMN1, that results in alternative splicing because the substitutiondisrupts recognition of the upstream 3′ splice site, in which exon 7 isfrequently skipped during precursor mRNA splicing. Consequently, SMN2encodes primarily the exon 7-skipped protein isoform (SMNA7), which isunstable, mislocalized, and only partially functional.

Methods and related single stranded oligonucleotides that are useful forselectively inducing expression of particular splice variants of SMN1 orSMN2 are provided herein. The methods are useful for controlling thelevels in a cell of particular SMN protein isoforms encoded by thesplice variants. In some cases, the methods are useful for inducingexpression of SMN proteins to levels sufficient to treat SMA. Forexample, according to some aspects of the invention methods are providedfor increasing expression of full-length SMN protein in a cell forpurposes of treating SMA. In some embodiments, the methods comprisedelivering to the cell a first single stranded oligonucleotidecomplementary with a PRC2-associated region of SMN1 or SMN2 and a secondsingle stranded oligonucleotide complementary with a splice controlsequence of a precursor mRNA of SMN1 or SMN2, in amounts sufficient toincrease expression of a mature mRNA of SMN1 or SMN2 that comprises (orincludes) exon 7 in the cell. Further aspects of the invention aredescribed in detailed herein.

Polycomb Repressive Complex 2 (PRC2)-Interacting RNAs

Aspects of the invention provided herein relate to the discovery ofpolycomb repressive complex 2 (PRC2)-interacting RNAs. Polycombrepressive complex 2 (PRC2) is a histone methyltransferase and a knownepigenetic regulator involved in silencing of genomic regions throughmethylation of histone H3. Among other functions, PRC2 interacts withlong noncoding RNAs (lncRNAs), such as RepA, Xist, and Tsix, to catalyzetrimethylation of histone H3-lysine27. PRC2 contains four subunits, Eed,Suz12, RbAp48, and Ezh2. Aspects of the invention relate to therecognition that single stranded oligonucleotides that bind toPRC2-associated regions of RNAs (e.g., lncRNAs) that are expressed fromwithin a genomic region that encompasses or that is in functionalproximity to the SMN1 or SMN2 gene can induce or enhance expression ofSMN1 or SMN2. In some embodiments, this upregulation is believed toresult from inhibition of PRC2 mediated repression of SMN1 or SMN2.

As used herein, the term “PRC2-associated region” refers to a region ofa nucleic acid that comprises or encodes a sequence of nucleotides thatinteract directly or indirectly with a component of PRC2. APRC2-associated region may be present in a RNA (e.g., a long non-codingRNA (lncRNA)) that interacts with a PRC2. A PRC2-associated region maybe present in a DNA that encodes an RNA that interacts with PRC2. Insome cases, the PRC2-associated region is equivalently referred to as aPRC2-interacting region.

In some embodiments, a PRC2-associated region is a region of an RNA thatcrosslinks to a component of PRC2 in response to in situ ultravioletirradiation of a cell that expresses the RNA, or a region of genomic DNAthat encodes that RNA region. In some embodiments, a PRC2-associatedregion is a region of an RNA that immunoprecipitates with an antibodythat targets a component of PRC2, or a region of genomic DNA thatencodes that RNA region. In some embodiments, a PRC2-associated regionis a region of an RNA that immunoprecipitates with an antibody thatbinds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted aboveare components of PRC2), or a region of genomic DNA that encodes thatRNA region.

In some embodiments, a PRC2-associated region is a region of an RNA thatis protected from nucleases (e.g., RNases) in an RNA-immunoprecipitationassay that employs an antibody that targets a component of PRC2, or aregion of genomic DNA that encodes that protected RNA region. In someembodiments, a PRC2-associated region is a region of an RNA that isprotected from nucleases (e.g., RNases) in an RNA-immunoprecipitationassay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4,or a region of genomic DNA that encodes that protected RNA region.

In some embodiments, a PRC2-associated region is a region of an RNAwithin which occur a relatively high frequency of sequence reads in asequencing reaction of products of an RNA-immunoprecipitation assay thatemploys an antibody that targets a component of PRC2, or a region ofgenomic DNA that encodes that RNA region. In some embodiments, aPRC2-associated region is a region of an RNA within which occur arelatively high frequency of sequence reads in a sequencing reaction ofproducts of an RNA-immunoprecipitation assay that employs an antibodythat binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region ofgenomic DNA that encodes that protected RNA region. In such embodiments,the PRC2-associated region may be referred to as a “peak.”

In some embodiments, a PRC2-associated region comprises a sequence of 40to 60 nucleotides that interact with PRC2 complex. In some embodiments,a PRC2-associated region comprises a sequence of 40 to 60 nucleotidesthat encode an RNA that interacts with PRC2. In some embodiments, aPRC2-associated region comprises a sequence of up to 5 kb in length thatcomprises a sequence (e.g., of 40 to 60 nucleotides) that interacts withPRC2. In some embodiments, a PRC2-associated region comprises a sequenceof up to 5 kb in length within which an RNA is encoded that has asequence (e.g., of 40 to 60 nucleotides) that is known to interact withPRC2. In some embodiments, a PRC2-associated region comprises a sequenceof about 4 kb in length that comprise a sequence (e.g., of 40 to 60nucleotides) that interacts with PRC2. In some embodiments, aPRC2-associated region comprises a sequence of about 4 kb in lengthwithin which an RNA is encoded that includes a sequence (e.g., of 40 to60 nucleotides) that is known to interact with PRC2. In someembodiments, a PRC2-associated region has a sequence as set forth in anyone of SEQ ID NOS: 9 to 29. In some embodiments, a PRC2-associatedregion has a sequence as set forth in any one of SEQ ID NOS: 24 to 29.

In some embodiments, single stranded oligonucleotides are provided thatspecifically bind to, or are complementary to, a PRC2-associated regionin a genomic region that encompasses or that is in proximity to the SMN1or SMN2 gene. In some embodiments, single stranded oligonucleotides areprovided that specifically bind to, or are complementary to, aPRC2-associated region that has a sequence as set forth in any one ofSEQ ID NOS: 9 to 29. In some embodiments, single strandedoligonucleotides are provided that specifically bind to, or arecomplementary to, a PRC2-associated region that has a sequence as setforth in any one of SEQ ID NOS: 9 to 29 combined with up to 2 kb, up to5 kb, or up to 10 kb of flanking sequences from a corresponding genomicregion to which these SEQ IDs map (e.g., in a human genome). In someembodiments, single stranded oligonucleotides have a sequence as setforth in any one of SEQ ID NOS: 30 to 13087. In some embodiments, singlestranded oligonucleotides have a sequence as set forth in Table 2. Insome embodiments, a PRC2 associated region of SMN1 or SMN2 against whicha single stranded oligonucleotide is complementary is selected from SEQID NOS: 24-29. In some embodiments, a single stranded oligonucleotidethat is complementary with a PRC2 associated region of SMN1 or SMN2comprises a sequence selected from SEQ ID NOS: 1158-1159, 1171,1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2546, 2656-2657,2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537, 6061,7327, 8330-13061, and 13062-13087. In some embodiments, a singlestranded oligonucleotide that is complementary with a PRC2 associatedregion of SMN1 or SMN2 comprises a sequence selected from 11395, 11394,10169, and 10170.

Without being bound by a theory of invention, these oligonucleotides areable to interfere with the binding of and function of PRC2, bypreventing recruitment of PRC2 to a specific chromosomal locus. Forexample, a single administration of single stranded oligonucleotidesdesigned to specifically bind a PRC2-associated region lncRNA can stablydisplace not only the lncRNA, but also the PRC2 that binds to thelncRNA, from binding chromatin. After displacement, the full complementof PRC2 is not recovered for up to 24 hours. Further, lncRNA can recruitPRC2 in a cis fashion, repressing gene expression at or near thespecific chromosomal locus from which the lncRNA was transcribed.

Methods of modulating gene expression are provided, in some embodiments,that may be carried out in vitro, ex vivo, or in vivo. It is understoodthat any reference to uses of compounds throughout the descriptioncontemplates use of the compound in preparation of a pharmaceuticalcomposition or medicament for use in the treatment of condition (e.g.,Spinal muscular atrophy) associated with decreased levels or activity ofSMN1 or SMN2. Thus, as one nonlimiting example, this aspect of theinvention includes use of such single stranded oligonucleotides in thepreparation of a medicament for use in the treatment of disease, whereinthe treatment involves upregulating expression of SMN1 or SMN2.

In further aspects of the invention, methods are provided for selectinga candidate oligonucleotide for activating expression of SMN1 or SMN2.The methods generally involve selecting as a candidate oligonucleotide,a single stranded oligonucleotide comprising a nucleotide sequence thatis complementary to a PRC2-associated region (e.g., a nucleotidesequence as set forth in any one of SEQ ID NOS: 9 to 29). In someembodiments, sets of oligonucleotides may be selected that are enriched(e.g., compared with a random selection of oligonucleotides) inoligonucleotides that activate expression of SMN1 or SMN2.

Single Stranded Oligonucleotides for Modulating Expression of SMN1 orSMN2

In one aspect of the invention, single stranded oligonucleotidescomplementary to the PRC2-associated regions are provided for modulatingexpression of SMN1 or SMN2 in a cell. In some embodiments, expression ofSMN1 or SMN2 is upregulated or increased. In some embodiments, singlestranded oligonucleotides complementary to these PRC2-associated regionsinhibit the interaction of PRC2 with long RNA transcripts such that geneexpression is upregulated or increased. In some embodiments, singlestranded oligonucleotides complementary to these PRC2-associated regionsinhibit the interaction of PRC2 with long RNA transcripts, resulting inreduced methylation of histone H3 and reduced gene inactivation, suchthat gene expression is upregulated or increased. In some embodiments,this interaction may be disrupted or inhibited due to a change in thestructure of the long RNA that prevents or reduces binding to PRC2. Theoligonucleotide may be selected using any of the methods disclosedherein for selecting a candidate oligonucleotide for activatingexpression of SMN1 or SMN2.

The single stranded oligonucleotide may comprise a region ofcomplementarity that is complementary with a PRC2-associated region of anucleotide sequence set forth in any one of SEQ ID NOS: 1 to 8. Theregion of complementarity of the single stranded oligonucleotide may becomplementary with at least 6, e.g., at least 7, at least 8, at least 9,at least 10, at least 15 or more consecutive nucleotides of thePRC2-associated region.

It should be appreciated that due the high homology between SMN1 andSMN2, single stranded oligonucleotides that are complementary with aPRC2-associated region of SMN1 are often also complementary with acorresponding PRC2-associated region of SMN2.

The PRC2-associated region may map to a position in a chromosome between50 kilobases upstream of a 5′-end of the SMN1 or SMN2 gene and 50kilobases downstream of a 3′-end of the SMN1 or SMN2 gene. ThePRC2-associated region may map to a position in a chromosome between 25kilobases upstream of a 5′-end of the SMN1 or SMN2 gene and 25 kilobasesdownstream of a 3′-end of the SMN1 or SMN2 gene. The PRC2-associatedregion may map to a position in a chromosome between 12 kilobasesupstream of a 5′-end of the SMN1 or SMN2 gene and 12 kilobasesdownstream of a 3′-end of the SMN1 or SMN2 gene. The PRC2-associatedregion may map to a position in a chromosome between 5 kilobasesupstream of a 5′-end of the SMN1 or SMN2 gene and 5 kilobases downstreamof a 3′-end of the SMN1 or SMN2 gene.

The genomic position of the selected PRC2-associated region relative tothe SMN1 or SMN2 gene may vary. For example, the PRC2-associated regionmay be upstream of the 5′ end of the SMN1 or SMN2 gene. ThePRC2-associated region may be downstream of the 3′ end of the SMN1 orSMN2 gene. The PRC2-associated region may be within an intron of theSMN1 or SMN2 gene. The PRC2-associated region may be within an exon ofthe SMN1 or SMN2 gene. The PRC2-associated region may traverse anintron-exon junction, a 5′-UTR-exon junction or a 3′-UTR-exon junctionof the SMN1 or SMN2 gene.

The single stranded oligonucleotide may comprise a sequence having theformula X-Y-Z, in which X is any nucleotide, Y is a nucleotide sequenceof 6 nucleotides in length that is not a human seed sequence of amicroRNA, and Z is a nucleotide sequence of varying length. In someembodiments X is the 5′ nucleotide of the oligonucleotide. In someembodiments, when X is anchored at the 5′ end of the oligonucleotide,the oligonucleotide does not have any nucleotides or nucleotide analogslinked 5′ to X. In some embodiments, other compounds such as peptides orsterols may be linked at the 5′ end in this embodiment as long as theyare not nucleotides or nucleotide analogs. In some embodiments, thesingle stranded oligonucleotide has a sequence 5′X-Y-Z and is 8-50nucleotides in length. Oligonucleotides that have these sequencecharacteristics are predicted to avoid the miRNA pathway. Therefore, insome embodiments, oligonucleotides having these sequence characteristicsare unlikely to have an unintended consequence of functioning in a cellas a miRNA molecule. The Y sequence may be a nucleotide sequence of 6nucleotides in length set forth in Table 1.

The single stranded oligonucleotide may have a sequence that does notcontain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 ormore, 6 or more consecutive guanosine nucleotides). In some embodiments,oligonucleotides having guanosine nucleotide stretches have increasednon-specific binding and/or off-target effects, compared witholigonucleotides that do not have guanosine nucleotide stretches.

The single stranded oligonucleotide may have a sequence that has lessthan a threshold level of sequence identity with every sequence ofnucleotides, of equivalent length, that map to a genomic positionencompassing or in proximity to an off-target gene. For example, anoligonucleotide may be designed to ensure that it does not have asequence that maps to genomic positions encompassing or in proximitywith all known genes (e.g., all known protein coding genes) other thanSMN1 or SMN2. In a similar embodiment, an oligonucleotide may bedesigned to ensure that it does not have a sequence that maps to anyother known PRC2-associated region, particularly PRC2-associated regionsthat are functionally related to any other known gene (e.g., any otherknown protein coding gene). In either case, the oligonucleotide isexpected to have a reduced likelihood of having off-target effects. Thethreshold level of sequence identity may be 50%, 60%, 70%, 80%, 85%,90%, 95%, 99% or 100% sequence identity.

The single stranded oligonucleotide may have a sequence that iscomplementary to a PRC2-associated region that encodes an RNA that formsa secondary structure comprising at least two single stranded loops. Inhas been discovered that, in some embodiments, oligonucleotides that arecomplementary to a PRC2-associated region that encodes an RNA that formsa secondary structure comprising one or more single stranded loops(e.g., at least two single stranded loops) have a greater likelihood ofbeing active (e.g., of being capable of activating or enhancingexpression of a target gene) than a randomly selected oligonucleotide.In some cases, the secondary structure may comprise a double strandedstem between the at least two single stranded loops. Accordingly, theregion of complementarity between the oligonucleotide and thePRC2-associated region may be at a location of the PRC2 associatedregion that encodes at least a portion of at least one of the loops. Insome cases, the region of complementarity between the oligonucleotideand the PRC2-associated region may be at a location of thePRC2-associated region that encodes at least a portion of at least twoof the loops. In some cases, the region of complementarity between theoligonucleotide and the PRC2-associated region may be at a location ofthe PRC2 associated region that encodes at least a portion of the doublestranded stem. In some embodiments, a PRC2-associated region (e.g., ofan lncRNA) is identified (e.g., using RIP-Seq methodology or informationderived therefrom). In some embodiments, the predicted secondarystructure RNA (e.g., lncRNA) containing the PRC2-associated region isdetermined using RNA secondary structure prediction algorithms, e.g.,RNAfold, mfold. In some embodiments, oligonucleotides are designed totarget a region of the RNA that forms a secondary structure comprisingone or more single stranded loop (e.g., at least two single strandedloops) structures which may comprise a double stranded stem between theat least two single stranded loops.

The single stranded oligonucleotide may have a sequence that is hasgreater than 30% G-C content, greater than 40% G-C content, greater than50% G-C content, greater than 60% G-C content, greater than 70% G-Ccontent, or greater than 80% G-C content. The single strandedoligonucleotide may have a sequence that has up to 100% G-C content, upto 95% G-C content, up to 90% G-C content, or up to 80% G-C content. Insome embodiments in which the oligonucleotide is 8 to 10 nucleotides inlength, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementarysequence of the PRC2-associated region are cytosine or guanosinenucleotides. In some embodiments, the sequence of the PRC2-associatedregion to which the single stranded oligonucleotide is complementarycomprises no more than 3 nucleotides selected from adenine and uracil.

The single stranded oligonucleotide may be complementary to a chromosomeof a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.)at a position that encompasses or that is in proximity to that species'homolog of SMN1 or SMN2. The single stranded oligonucleotide may becomplementary to a human genomic region encompassing or in proximity tothe SMN1 or SMN2 gene and also be complementary to a mouse genomicregion encompassing or in proximity to the mouse homolog of SMN1 orSMN2. For example, the single stranded oligonucleotide may becomplementary to a sequence as set forth in SEQ ID NO: 1, 2, 4, or 5,which is a human genomic region encompassing or in proximity to the SMN1or SMN2 gene, and also be complementary to a sequence as set forth inSEQ ID NO:7 or 8, which is a mouse genomic region encompassing or inproximity to the mouse homolog of the SMN1 or SMN2 gene.Oligonucleotides having these characteristics may be tested in vivo orin vitro for efficacy in multiple species (e.g., human and mouse). Thisapproach also facilitates development of clinical candidates fortreating human disease by selecting a species in which an appropriateanimal exists for the disease.

In some embodiments, the region of complementarity of the singlestranded oligonucleotide is complementary with at least 8 to 15, 8 to30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of aPRC2-associated region. In some embodiments, the region ofcomplementarity is complementary with at least 8 consecutive nucleotidesof a PRC2-associated region. In some embodiments the sequence of thesingle stranded oligonucleotide is based on an RNA sequence that bindsto PRC2, or a portion thereof, said portion having a length of from 5 to40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, orabout 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.Complementary, as the term is used in the art, refers to the capacityfor precise pairing between two nucleotides. For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position ofPRC2-associated region, then the single stranded nucleotide andPRC2-associated region are considered to be complementary to each otherat that position. The single stranded nucleotide and PRC2-associatedregion are complementary to each other when a sufficient number ofcorresponding positions in each molecule are occupied by nucleotidesthat can hydrogen bond with each other through their bases. Thus,“complementary” is a term which is used to indicate a sufficient degreeof complementarity or precise pairing such that stable and specificbinding occurs between the single stranded nucleotide andPRC2-associated region. For example, if a base at one position of asingle stranded nucleotide is capable of hydrogen bonding with a base atthe corresponding position of a PRC2-associated region, then the basesare considered to be complementary to each other at that position. 100%complementarity is not required.

The single stranded oligonucleotide may be at least 80% complementary to(optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% complementary to) the consecutive nucleotides of aPRC2-associated region. In some embodiments the single strandedoligonucleotide may contain 1, 2 or 3 base mismatches compared to theportion of the consecutive nucleotides of a PRC2-associated region. Insome embodiments the single stranded oligonucleotide may have up to 3mismatches over 15 bases, or up to 2 mismatches over 10 bases.

It is understood in the art that a complementary nucleotide sequenceneed not be 100% complementary to that of its target to be specificallyhybridizable. In some embodiments, a complementary nucleic acid sequencefor purposes of the present disclosure is specifically hybridizable whenbinding of the sequence to the target molecule (e.g., lncRNA) interfereswith the normal function of the target (e.g., lncRNA) to cause a loss ofactivity (e.g., inhibiting PRC2-associated repression with consequentup-regulation of gene expression) and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the sequence tonon-target sequences under conditions in which avoidance of non-specificbinding is desired, e.g., under physiological conditions in the case ofin vivo assays or therapeutic treatment, and in the case of in vitroassays, under conditions in which the assays are performed undersuitable conditions of stringency.

In some embodiments, the single stranded oligonucleotide is 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferredembodiment, the oligonucleotide is 8 to 30 nucleotides in length.

In some embodiments, the PRC2-associated region occurs on the same DNAstrand as a gene sequence (sense). In some embodiments, thePRC2-associated region occurs on the opposite DNA strand as a genesequence (anti-sense). Oligonucleotides complementary to aPRC2-associated region can bind either sense or anti-sense sequences.Base pairings may include both canonical Watson-Crick base pairing andnon-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteenbase pairing). It is understood that for complementary base pairings,adenosine-type bases (A) are complementary to thymidine-type bases (T)or uracil-type bases (U), that cytosine-type bases (C) are complementaryto guanosine-type bases (G), and that universal bases such as3-nitropyrrole or 5-nitroindole can hybridize to and are consideredcomplementary to any A, C, U, or T. Inosine (1) has also been consideredin the art to be a universal base and is considered complementary to anyA, C, U or T.

In some embodiments, any one or more thymidine (T) nucleotides (ormodified nucleotide thereof) or uridine (U) nucleotides (or a modifiednucleotide thereof) in a sequence provided herein, including a sequenceprovided in the sequence listing, may be replaced with any othernucleotide suitable for base pairing (e.g., via a Watson-Crick basepair) with an adenosine nucleotide. In some embodiments, any one or morethymidine (T) nucleotides (or modified nucleotide thereof) or uridine(U) nucleotides (or a modified nucleotide thereof) in a sequenceprovided herein, including a sequence provided in the sequence listing,may be suitably replaced with a different pyrimidine nucleotide or viceversa. In some embodiments, any one or more thymidine (T) nucleotides(or modified nucleotide thereof) in a sequence provided herein,including a sequence provided in the sequence listing, may be suitablyreplaced with a uridine (U) nucleotide (or a modified nucleotidethereof) or vice versa. In some embodiments, GC content of the singlestranded oligonucleotide is preferably between about 30-60%. Contiguousruns of three or more Gs or Cs may not be preferable in someembodiments. Accordingly, in some embodiments, the oligonucleotide doesnot comprise a stretch of three or more guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide specificallybinds to, or is complementary to an RNA that is encoded in a genome(e.g., a human genome) as a single contiguous transcript (e.g., anon-spliced RNA). In some embodiments, the single strandedoligonucleotide specifically binds to, or is complementary to an RNAthat is encoded in a genome (e.g., a human genome), in which thedistance in the genome between the 5′end of the coding region of the RNAand the 3′ end of the coding region of the RNA is less than 1 kb, lessthan 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.

It is to be understood that any oligonucleotide provided herein can beexcluded.

In some embodiments, single stranded oligonucleotides disclosed hereinmay increase expression of mRNA corresponding to the gene by at leastabout 50% (i.e. 150% of normal or 1.5 fold), or by about 2 fold to about5 fold. In some embodiments, expression may be increased by at leastabout 15 fold, 20 fold, 30 fold, 40 fold, 50 fold or 100 fold, or anyrange between any of the foregoing numbers. It has also been found thatincreased mRNA expression has been shown to correlate to increasedprotein expression.

In some or any of the embodiments of the oligonucleotides describedherein, or processes for designing or synthesizing them, theoligonucleotides will upregulate gene expression and may specificallybind or specifically hybridize or be complementary to the PRC2 bindingRNA that is transcribed from the same strand as a protein codingreference gene. The oligonucleotide may bind to a region of the PRC2binding RNA that originates within or overlaps an intron, exon, intronexon junction, 5′ UTR, 3′ UTR, a translation initiation region, or atranslation termination region of a protein coding sense strand of areference gene (refGene).

In some or any of the embodiments of oligonucleotides described herein,or processes for designing or synthesizing them, the oligonucleotideswill upregulate gene expression and may specifically bind orspecifically hybridize or be complementary to a PRC2 binding RNA thattranscribed from the opposite strand (the antisense strand) of a proteincoding reference gene. The oligonucleotide may bind to a region of thePRC2 binding RNA that originates within or overlaps an intron, exon,intron exon junction, 5′ UTR, 3′ UTR, a translation initiation region,or a translation termination region of a protein coding antisense strandof a reference gene.

The oligonucleotides described herein may be modified, e.g., comprise amodified sugar moiety, a modified internucleoside linkage, a modifiednucleotide and/or combinations thereof. In addition, theoligonucleotides can exhibit one or more of the following properties: donot induce substantial cleavage or degradation of the target RNA; do notcause substantially complete cleavage or degradation of the target RNA;do not activate the RNAse H pathway; do not activate RISC; do notrecruit any Argonaute family protein; are not cleaved by Dicer; do notmediate alternative splicing; are not immune stimulatory; are nucleaseresistant; have improved cell uptake compared to unmodifiedoligonucleotides; are not toxic to cells or mammals; may have improvedendosomal exit; do interfere with interaction of lncRNA with PRC2,preferably the Ezh2 subunit but optionally the Suz12, Eed, RbAp46/48subunits or accessory factors such as Jarid2; do decrease histone H3lysine27 methylation and/or do upregulate gene expression.

Oligonucleotides that are designed to interact with RNA to modulate geneexpression are a distinct subset of base sequences from those that aredesigned to bind a DNA target (e.g., are complementary to the underlyinggenomic DNA sequence from which the RNA is transcribed).

Splice Switching Oligonucleotides Aspects of the invention providestrategies for targeting SMN1 or SMN2 precursor mRNA to affect splicingto minimize exon skipping. Accordingly, aspects of the invention providetherapeutic compounds useful for the treatment of SMA. In someembodiments, oligonucleotides, referred to herein as “splice switchingoligonucleotides” are provided that modulate SMN2 splicing. Methods andrelated compositions, compounds, and kits are provided, in someembodiments, that are useful for increasing expression of full-length.SMN protein in a cell. The methods generally involve delivering to acell a first single stranded oligonucleotide complementary with at least8 consecutive nucleotides of a PRC2-associated region of SMN2 and asecond single stranded oligonucleotide complementary with a splicecontrol sequence of a precursor mRNA of SMN2, in amounts sufficient toincrease expression of a mature mRNA of SMN2 that comprises (orincludes) exon 7 in the cell. Any of the single strandedoligonucleotides that are complementary with at least 8 consecutivenucleotides of a PRC2-associated region of SMN1 or SMN2 may be used. Itshould be appreciated that single stranded oligonucleotides that arecomplementary with a splice control sequence may alternatively bereferred herein, as splice switching oligonucleotides.

Splice switching oligonucleotides typically comprise a sequencecomplementary to a splice control sequence (e.g., a intronic splicingsilencer sequence) of a precursor mRNA, and are capable of binding toand affecting processing of the precursor mRNA. Splice switchingoligonucleotides may be complementary with a region of an exon, a regionof an intron or an intron/exon junction. In some embodiments, the splicecontrol sequence comprises the sequence: CAGCAUUAUGAAAG (SEQ ID NO:13100) or a portion thereof. In some embodiments, the splice controlsequence comprises at least one hnRNAP binding sequence. In someembodiments, splice switching oligonucleotides that target SMN1 or SMN2function based on the premise that there is a competition between the 3′splice sites of exons 7 and 8 for pairing with the 5′ splice site ofexon 6, so impairing the recognition of the 3′ splice site of exon 8favors exon 7 inclusion. In some embodiments, splice switchingoligonucleotides are provided that promote SMN2 exon 7 inclusion andfull-length SMN protein expression, in which the oligonucleotides arecomplementary to the intron 7/exon 8 junction. In some embodiments,splice switching oligonucleotide are composed of a segment complementaryto an exon of SMN1 or SMN2 (e.g., exon 7). In some embodiments, spliceswitching oligonucleotides comprise a tail (e.g., a non-complementarytail) consisting of RNA sequences with binding motifs recognized by aserine/arginine-rich (SR) protein. In some embodiments, splice switchingoligonucleotides are complementary (at least partially) with an intronicsplicing silencer (ISS). In some embodiments, the ISS is in intron 6 orintron 7 of SMN1 or SMN2. In some embodiments, splice switchingoligonucleotides comprise an antisense moiety complementary to a targetexon or intron (e.g., of SMN1 or SMN2) and a minimal RS domain peptidesimilar to the splicing activation domain of SR proteins. In someembodiments, the splice switching oligonucleotide is 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50 or more nucleotides in length. In one embodiment, theoligonucleotide is 8 to 30 nucleotides in length.

Linkers

Any of the oligonucleotides disclosed herein may be linked to one ormore other oligonucleotides disclosed herein by a linker, e.g., acleavable linker. Accordingly, in some embodiments, compounds areprovided that comprise a single stranded oligonucleotide complementarywith a PRC2-associated region of a gene that is linked via a linker to asingle stranded oligonucleotide complementary to a splice controlsequence of a precursor mRNA of the gene. In some embodiments, compoundsare provided that have the general formula A-B-C, in which A is a singlestranded oligonucleotide complementary with a PRC2-associated region ofa gene, B is a linker, and C is a single stranded oligonucleotidecomplementary to a splice control sequence of a precursor mRNA of thegene. In some embodiments, linker B comprises an oligonucleotide,peptide, low pH labile bond, or disulfide bond. In some embodiments, thecompounds is orientated as 5′-A-B-C-3′. In some embodiments, thecompound is orientated as 3′-A-B-C-5′. In some embodiments, where B isan oligonucleotide, the 3′ end of A is linked to the 5′ end of B, andthe 3′ end of B is linked to 5′ end of C. In some embodiments, where Bis an oligonucleotide, the 5′ end of A is linked to the 3′ end of B, andthe 5′ end of B is linked to 3′ end of C. In some embodiments, where Bis an oligonucleotide, the 5′ end of A is linked to the 5′ end of B,and/or the 3′ end of B is linked to the 3′ end of C. In someembodiments, where B is an oligonucleotide, the 3′ end of A is linked tothe 3′ end of B, and/or the 5′ end of B is linked to the 5′ end of C.

The term “linker” generally refers to a chemical moiety that is capableof covalently linking two or more oligonucleotides. In some embodiments,at least one bond comprised or contained within the linker is capable ofbeing cleaved (e.g., in a biological context, such as in a mammalianextract, such as an endosomal extract), such that at least twooligonucleotides are no longer covalently linked to one another afterbond cleavage. It will be appreciated that, in some embodiments, aprovided linker may include a region that is non-cleavable, as long asthe linker also comprises at least one bond that is cleavable.

In some embodiments, the linker comprises a polypeptide that is moresusceptible to cleavage by an endopeptidase in the mammalian extractthan the oligonucleotides. The endopeptidase may be a trypsin,chymotrypsin, elastase, thermolysin, pepsin, or endopeptidase V8. Theendopeptidase may be a cathepsin B, cathepsin D, cathepsin L, cathepsinC, papain, cathepsin S or endosomal acidic insulinase. For example, thelinker comprise a peptide having an amino acid sequence selected from:ALAL, APISFFELG, FL, GFN, R/KXX, GRWHTVGLRWE, YL, GF, and FF, in which Xis any amino acid.

In some embodiments, the linker comprises the formula—(CH₂)_(n)S—S(CH₂)_(m)—, wherein n and m are independently integers from0 to 10.

In some embodiments, the linker may comprise an oligonucleotide that ismore susceptible to cleavage by an endonuclease in the mammalian extractthan the oligonucleotides. The linker may have a nucleotide sequencecomprising from 1 to 10 thymidines or uridines. The linker may have anucleotide sequence comprising deoxyribonucleotides linked throughphosphodiester internucleotide linkages. The linker may have anucleotide sequence comprising from 1 to 10 thymidines or uridineslinked through phosphodiester internucleotide linkages. The linker mayhave a nucleotide sequence comprising from 1 to 10 thymidines oruridines linked through phosphorothioate internucleotide linkages.

In some embodiments, at least one linker is 2-fold, 3-fold, 4-fold,5-fold, 10-fold or more sensitive to enzymatic cleavage in the presenceof a mammalian extract than at least two oligonucleotides. It should beappreciated that different linkers can be designed to be cleaved atdifferent rates and/or by different enzymes in compounds comprising twoor more linkers. Similarly different linkers can be designed to besensitive to cleavage in different tissues, cells or subcellularcompartments in compounds comprising two or more linkers. This canadvantageously permit compounds to have oligonucleotides that arereleased from compounds at different rates, by different enzymes, or indifferent tissues, cells or subcellular compartments thereby controllingrelease of the monomeric oligonucleotides to a desired in vivo locationor at a desired time following administration.

In certain embodiments, linkers are stable (e.g., more stable than theoligonucleotides they link together) in plasma, blood or serum which arericher in exonucleases, and less stable in the intracellularenvironments which are relatively rich in endonucleases. In someembodiments, a linker is considered “non-cleavable” if the linker'shalf-life is at least 24, or 28, 32, 36, 48, 72, 96 hours or longerunder the conditions described here, such as in liver homogenates.Conversely, in some embodiments, a linker is considered “cleavable” ifthe half-life of the linker is at most 10, or 8, 6, 5 hours or shorter.

In some embodiments, the linker is a nuclease-cleavable oligonucleotidelinker. In some embodiments, the nuclease-cleavable linker contains oneor more phosphodiester bonds in the oligonucleotide backbone. Forexample, the linker may contain a single phosphodiester bridge or 2, 3,4, 5, 6, 7 or more phosphodiester linkages, for example as a string of1-10 deoxynucleotides, e.g., dT, or ribonucleotides, e.g., rU, in thecase of RNA linkers. In the case of dT or other DNA nucleotides dN inthe linker, in certain embodiments the cleavable linker contains one ormore phosphodiester linkages. In other embodiments, in the case of rU orother RNA nucleotides rN, the cleavable linker may consist ofphosphorothioate linkages only. In contrast to phosphorothioate-linkeddeoxynucleotides, which in some embodiments are cleaved relativelyslowly by nucleases (thus termed “noncleavable”),phosphorothioate-linked rU undergoes relatively rapid cleavage byribonucleases and therefore is considered cleavable herein in someembodiments. It is also possible to combine dN and rN into the linkerregion, which are connected by phosphodiester or phosphorothioatelinkages. In other embodiments, the linker can also contain chemicallymodified nucleotides, which are still cleavable by nucleases, such as,e.g., 2′-O-modified analogs. In particular, 2′-O-methyl or 2′-fluoronucleotides can be combined with each other or with dN or rNnucleotides. Generally, in the case of nucleotide linkers, the linker isa part of the compound that is usually not complementary to a target,although it could be. This is because the linker is generally cleavedprior to action of the oligonucleotides on the target, and therefore,the linker identity with respect to a target is inconsequential.Accordingly, in some embodiments, a linker is an (oligo)nucleotidelinker that is not complementary to any of the targets against which theoligonucleotides are designed.

In some embodiments, the cleavable linker is an oligonucleotide linkerthat contains a continuous stretch of deliberately introduced Rpphosphorothioate stereoisomers (e.g., 4, 5, 6, 7 or longer stretches).The Rp stereoisoform, unlike Sp isoform, is known to be susceptible tonuclease cleavage (Krieg et al., 2003, Oligonucleotides, 13:491-499).Such a linker would not include a racemic mix of PS linkagedoligonucleotides since the mixed linkages are relatively stable and arenot likely to contain long stretches of the Rp stereoisomers, andtherefore, considered “non-cleavable” herein. Thus, in some embodiments,a linker comprises a stretch of 4, 5, 6, 7 or more phosphorothioatednucleotides, wherein the stretch does not contain a substantial amountor any of the Sp stereoisoform. The amount could be consideredsubstantial if it exceeds 10% on a per-mole basis.

In some embodiments, the linker is a non-nucleotide linker, for example,a single phosphodiester bridge. Another example of such cleavablelinkers is a chemical group comprising a disulfide bond, for example,—(CH₂)_(n)S—S(CH₂)_(m)—, wherein n and m are integers from 0 to 10. Inillustrative embodiments, n=m=6. Additional examples of non-nucleotidelinkers are described below.

The linker can be designed so as to undergo a chemical or enzymaticcleavage reaction. Chemical reactions involve, for example, cleavage inacidic environments (e.g., endosomes), reductive cleavage (e.g.,cytosolic cleavage) or oxidative cleavage (e.g., in liver microsomes).The cleavage reaction can also be initiated by a rearrangement reaction.Enzymatic reactions can include reactions mediated by nucleases,peptidases, proteases, phosphatases, oxidases, reductases, etc. Forexample, a linker can be pH-sensitive, cathepsin-sensitive, orpredominantly cleaved in endosomes and/or cytosol.

In some embodiments, the linker comprises a peptide. In certainembodiments, the linker comprises a peptide which includes a sequencethat is cleavable by an endopeptidase. In addition to the cleavablepeptide sequence, the linker may comprise additional amino acid residuesand/or non-peptide chemical moieties, such as an alkyl chain. In certainembodiments, the linker comprises Ala-Leu-Ala-Leu, which is a substratefor cathepsin B. See, for example, themaleimidocaproyl-Arg-Arg-Ala-Leu-Ala-Leu linkers described in Schmid etal, Bioconjugate Chem 2007, 18, 702-716. In certain embodiments, acathepsin B-cleavable linker is cleaved in tumor cells. In certainembodiments, the linker comprises Ala-Pro-Ile-Ser-Phe-Phe-Glu-Leu-Gly,which is a substrate for cathepsins D, L, and B (see, for example,Fischer et al, Chembiochem 2006, 7, 1428-1434). In certain embodiments,a cathepsin-cleavable linker is cleaved in HeLA cells. In someembodiments, the linker comprises Phe-Lys, which is a substrate forcathepsin B. For example, in certain embodiments, the linker comprisesPhe-Lys-p-aminobenzoic acid (PABA). See, e.g., themaleimidocaproyl-Phe-Lys-PABA linker described in Walker et al, Bioorg.Med. Chem. Lett. 2002, 12, 217-219. In certain embodiments, the linkercomprises Gly-Phe-2-naphthylamide, which is a substrate for cathepsin C(see, for example, Berg et al. Biochem. J. 1994, 300, 229-235). Incertain embodiments, a cathepsin C-cleavable linker is cleaved inhepatocytes. In some embodiments, the linker comprises a cathepsin Scleavage site. For example, in some embodiments, the linker comprisesGly-Arg-Trp-His-Thr-Val-Gly-Leu-Arg-Trp-Glu,Gly-Arg-Trp-Pro-Pro-Met-Gly-Leu-Pro-Trp-Glu, orGly-Arg-Trp-His-Pro-Met-Gly-Ala-Pro-Trp-Glu, for example, as describedin Lutzner et al, J. Biol. Chem. 2008, 283, 36185-36194. In certainembodiments, a cathepsin S-cleavable linker is cleaved in antigenpresenting cells. In some embodiments, the linker comprises a papaincleavage site. Papain typically cleaves a peptide having the sequence-R/K-X-X (see Chapman et al, Annu. Rev. Physiol 1997, 59, 63-88). Incertain embodiments, a papain-cleavable linker is cleaved in endosomes.In some embodiments, the linker comprises an endosomal acidic insulinasecleavage site. For example, in some embodiments, the linker comprisesTyr-Leu, Gly-Phe, or Phe-Phe (see, e.g., Authier et al, FEBS Lett. 1996,389, 55-60). In certain embodiments, an endosomal acidicinsulinase-cleavable linker is cleaved in hepatic cells.

In some embodiments, the linker is pH sensitive. In certain embodiments,the linker comprises a low pH-labile bond. As used herein, a low-pHlabile bond is a bond that is selectively broken under acidic conditions(pH<7). Such bonds may also be termed endosomally labile bonds, becausecell endosomes and lysosomes have a pH less than 7. For example, incertain embodiments, the linker comprises an amine, an imine, an ester,a benzoic imine, an amino ester, a diortho ester, a polyphosphoester, apolyphosphazene, an acetal, a vinyl ether, a hydrazone, anazidomethyl-methylmaleic anhydride, a thiopropionate, a maskedendosomolytic agent or a citraconyl group.

In certain embodiments, the linker comprises a low pH-labile bondselected from the following: ketals that are labile in acidicenvironments (e.g., pH less than 7, greater than about 4) to form a dioland a ketone; acetals that are labile in acidic environments (e.g., pHless than 7, greater than about 4) to form a diol and an aldehyde;imines or iminiums that are labile in acidic environments (e.g., pH lessthan 7, greater than about 4) to form an amine and an aldehyde or aketone; silicon-oxygen-carbon linkages that are labile under acidiccondition; silicon-nitrogne (silazane) linkages; silicon-carbon linkages(e.g., arylsilanes, vinylsilanes, and allylsilanes); maleamates (amidebonds synthesized from maleic anhydride derivatives and amines); orthoesters; hydrazones; activated carboxylic acid derivatives (e.g., esters,amides) designed to undergo acid catalyzed hydrolysis); or vinyl ethers.Further examples may be found in International Patent Appln. Pub. No. WO2008/022309, entitled POLYCONJUGATES FOR IN VIVO DELIVERY OFPOLYNUCLEOTIDES, the contents of which are incorporated herein byreference.

In some embodiments, the linker comprises a masked endosomolytic agent.Endosomolytic polymers are polymers that, in response to a change in pH,are able to cause disruption or lysis of an endosome or provide forescape of a normally membrane-impermeable compound, such as apolynucleotide or protein, from a cellular internal membrane-enclosedvesicle, such as an endosome or lysosome. A subset of endosomolyticcompounds is fusogenic compounds, including fusogenic peptides.Fusogenic peptides can facilitate endosomal release of agents such asoligomeric compounds to the cytoplasm. See, for example, US PatentApplication Publication Nos. 20040198687, 20080281041, 20080152661, and20090023890, which are incorporated herein by reference.

The linker can also be designed to undergo an organ/tissue-specificcleavage. For example, for certain targets, which are expressed inmultiple tissues, only the knock-down in liver may be desirable, asknock-down in other organs may lead to undesired side effects. Thus,linkers susceptible to liver-specific enzymes, such as pyrrolase (TPO)and glucose-6-phosphatase (G-6-Pase), can be engineered, so as to limitthe antisense effect to the liver mainly. Alternatively, linkers notsusceptible to liver enzymes but susceptible to kidney-specific enzymes,such as gamma-glutamyltranspeptidase, can be engineered, so that theantisense effect is limited to the kidneys mainly. Analogously,intestine-specific peptidases cleaving Phe-Ala and Leu-Ala could beconsidered for orally administered multimeric oligonucleotides.Similarly, by placing an enzyme recognition site into the linker, whichis recognized by an enzyme over-expressed in tumors, such as plasmin(e.g., PHEA-D-Val-Leu-Lys recognition site), tumor-specific knock-downshould be feasible. By selecting the right enzyme recognition site inthe linker, specific cleavage and knock-down should be achievable inmany organs. In addition, the linker can also contain a targetingsignal, such as N-acetyl galactosamine for liver targeting, or folate,vitamin A or RGD-peptide in the case of tumor or activated macrophagetargeting. Accordingly, in some embodiments, the cleavable linker isorgan- or tissue-specific, for example, liver-specific, kidney-specific,intestine-specific, etc.

The oligonucleotides can be linked through any part of the individualoligonucleotide, e.g., via the phosphate, the sugar (e.g., ribose,deoxyribose), or the nucleobase. In certain embodiments, when linkingtwo oligonucleotides together, the linker can be attached e.g. to the5′-end of the first oligonucleotide and the 3′-end of the secondnucleotide, to the 5′-end of the first oligonucleotide and the 5′end ofthe second nucleotide, to the 3′-end of the first oligonucleotide andthe 3′-end of the second nucleotide. In other embodiments, when linkingtwo oligonucleotides together, the linker can attach internal residuesof each oligonucleotides, e.g., via a modified nucleobase. One ofordinary skill in the art will understand that many such permutationsare available for multimers. Further examples of appropriate linkers aswell as methods for producing compounds having such linkers aredisclosed in International Patent Application Number, PCT/US2012/05535,entitled MULTIMERIC OLIGONUCLEOTIDE COMPOUNDS the contents of whichrelating to linkers and related chemistries are incorporated herein byreferenced in its entirety.

Methods for Selecting Candidate Oligonucleotides for ActivatingExpression of SMN1 or SMN2

Methods are provided herein for selecting a candidate oligonucleotidefor activating or enhancing expression of SMN1 or SMN2. The targetselection methods may generally involve steps for selecting singlestranded oligonucleotides having any of the structural and functionalcharacteristics disclosed herein. Typically, the methods involve one ormore steps aimed at identifying oligonucleotides that target aPRC2-associated region that is functionally related to SMN1 or SMN2, forexample a PRC2-associated region of a lncRNA that regulates expressionof SMN1 or SMN2 by facilitating (e.g., in a cis-regulatory manner) therecruitment of PRC2 to the SMN1 or SMN2 gene. Such oligonucleotides areexpected to be candidates for activating expression of SMN1 or SMN2because of their ability to hybridize with the PRC2-associated region ofa nucleic acid (e.g., a lncRNA). In some embodiments, this hybridizationevent is understood to disrupt interaction of PRC2 with the nucleic acid(e.g., a lncRNA) and as a result disrupt recruitment of PRC2 and itsassociated co-repressors (e.g., chromatin remodeling factors) to theSMN1 or SMN2 gene locus.

Methods of selecting a candidate oligonucleotide may involve selecting aPRC2-associated region (e.g., a nucleotide sequence as set forth in anyone of SEQ ID NOS: 9 to 29) that maps to a chromosomal positionencompassing or in proximity to the SMN1 or SMN2 gene (e.g., achromosomal position having a sequence as set forth in any one of SEQ IDNOS: 1 to 8). The PRC2-associated region may map to the strand of thechromosome comprising the sense strand of the SMN1 or SMN2 gene, inwhich case the candidate oligonucleotide is complementary to the sensestrand of the SMN1 or SMN2 gene (i.e., is antisense to the SMN1 or SMN2gene). Alternatively, the PRC2-associated region may map to the strandof the first chromosome comprising the antisense strand of the SMN1 orSMN2 gene, in which case the oligonucleotide is complementary to theantisense strand (the template strand) of the SMN1 or SMN2 gene (i.e.,is sense to the SMN1 or SMN2 gene).

Methods for selecting a set of candidate oligonucleotides that isenriched in oligonucleotides that activate expression of SMN1 or SMN2may involve selecting one or more PRC2-associated regions that map to achromosomal position that encompasses or that is in proximity to theSMN1 or SMN2 gene and selecting a set of oligonucleotides, in which eacholigonucleotide in the set comprises a nucleotide sequence that iscomplementary with the one or more PRC2-associated regions. As usedherein, the phrase, “a set of oligonucleotides that is enriched inoligonucleotides that activate expression of” refers to a set ofoligonucleotides that has a greater number of oligonucleotides thatactivate expression of a target gene (e.g., SMN1 or SMN2) compared witha random selection of oligonucleotides of the same physicochemicalproperties (e.g., the same GC content, T_(m), length etc.) as theenriched set.

Where the design and/or synthesis of a single stranded oligonucleotideinvolves design and/or synthesis of a sequence that is complementary toa nucleic acid or PRC2-associated region described by such sequenceinformation, the skilled person is readily able to determine thecomplementary sequence, e.g., through understanding of Watson Crick basepairing rules which form part of the common general knowledge in thefield.

In some embodiments design and/or synthesis of a single strandedoligonucleotide involves manufacture of an oligonucleotide from startingmaterials by techniques known to those of skill in the art, where thesynthesis may be based on a sequence of a PRC2-associated region, orportion thereof.

Methods of design and/or synthesis of a single stranded oligonucleotidemay involve one or more of the steps of: Identifying and/or selectingPRC2-associated region; Designing a nucleic acid sequence having adesired degree of sequence identity or complementarity to aPRC2-associated region or a portion thereof;

Synthesizing a single stranded oligonucleotide to the designed sequence;

Purifying the synthesized single stranded oligonucleotide; and

Optionally mixing the synthesized single stranded oligonucleotide withat least one pharmaceutically acceptable diluent, carrier or excipientto form a pharmaceutical composition or medicament.

Single stranded oligonucleotides so designed and/or synthesized may beuseful in method of modulating gene expression as described herein.

Preferably, single stranded oligonucleotides of the invention aresynthesized chemically. Oligonucleotides used to practice this inventioncan be synthesized in vitro by well-known chemical synthesis techniques.

Oligonucleotides of the invention can be stabilized against nucleolyticdegradation such as by the incorporation of a modification, e.g., anucleotide modification. For example, nucleic acid sequences of theinvention include a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA).

As another example, the nucleic acid sequence can include at least one2′-O-methyl-modified nucleotide, and in some embodiments, all of thenucleotides include a 2′-O-methyl modification. In some embodiments, thenucleic acids are “locked,” i.e., comprise nucleic acid analogues inwhich the ribose ring is “locked” by a methylene bridge connecting the2′-O atom and the 4′-C atom.

It is understood that any of the modified chemistries or formats ofsingle stranded oligonucleotides described herein can be combined witheach other, and that one, two, three, four, five, or more differenttypes of modifications can be included within the same molecule.

In some embodiments, the method may further comprise the steps ofamplifying the synthesized single stranded oligonucleotide, and/orpurifying the single stranded oligonucleotide (or amplified singlestranded oligonucleotide), and/or sequencing the single strandedoligonucleotide so obtained.

As such, the process of preparing a single stranded oligonucleotide maybe a process that is for use in the manufacture of a pharmaceuticalcomposition or medicament for use in the treatment of disease,optionally wherein the treatment involves modulating expression of agene associated with a PRC2-associated region.

In the methods described above a PRC2-associated region may be, or havebeen, identified, or obtained, by a method that involves identifying RNAthat binds to PRC2.

Such methods may involve the following steps: providing a samplecontaining nuclear ribonucleic acids, contacting the sample with anagent that binds specifically to PRC2 or a subunit thereof, allowingcomplexes to form between the agent and protein in the sample,partitioning the complexes, synthesizing nucleic acid that iscomplementary to nucleic acid present in the complexes.

Where the single stranded oligonucleotide is based on a PRC2-associatedregion, or a portion of such a sequence, it may be based on informationabout that sequence, e.g., sequence information available in written orelectronic form, which may include sequence information contained inpublicly available scientific publications or sequence databases.

Nucleotide Analogues

In some embodiments, the oligonucleotide may comprise at least oneribonucleotide, at least one deoxyribonucleotide, and/or at least onebridged nucleotide. In some embodiments, the oligonucleotide maycomprise a bridged nucleotide, such as a locked nucleic acid (LNA)nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridgednucleic acid (ENA) nucleotide. Examples of such nucleotides aredisclosed herein and known in the art. In some embodiments, theoligonucleotide comprises a nucleotide analog disclosed in one of thefollowing United States patent or patent Application Publications: U.S.Pat. Nos. 7,399,845, 7,741,457, 8,022,193, 7,569,686, 7,335,765,7,314,923, 7,335,765, and 7,816,333, US 20110009471, the entire contentsof each of which are incorporated herein by reference for all purposes.The oligonucleotide may have one or more 2′ O-methyl nucleotides. Theoligonucleotide may consist entirely of 2′ O-methyl nucleotides.

Often the single stranded oligonucleotide has one or more nucleotideanalogues. For example, the single stranded oligonucleotide may have atleast one nucleotide analogue that results in an increase in T_(m) ofthe oligonucleotide in a range of P° C., 2° C., 3° C., 4° C., or 5° C.compared with an oligonucleotide that does not have the at least onenucleotide analogue. The single stranded oligonucleotide may have aplurality of nucleotide analogues that results in a total increase inT_(m), of the oligonucleotide in a range of 2° C., 3° C., 4° C., 5° C.,6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35°C., 40° C., 45° C. or more compared with an oligonucleotide that doesnot have the nucleotide analogue.

The oligonucleotide may be of up to 50 nucleotides in length in which 2to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide arenucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotidesin length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19,2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide arenucleotide analogues.

The oligonucleotide may be of 8 to 15 nucleotides in length in which 2to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 1, 2 to 12,2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotideanalogues. Optionally, the oligonucleotides may have every nucleotideexcept 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.

The oligonucleotide may consist entirely of bridged nucleotides (e.g.,LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotidemay comprise alternating deoxyribonucleotides and2′-fluoro-deoxyribonucleotides. The oligonucleotide may comprisealternating deoxyribonucleotides and 2′-O-methyl nucleotides. Theoligonucleotide may comprise alternating deoxyribonucleotides and ENAnucleotide analogues. The oligonucleotide may comprise alternatingdeoxyribonucleotides and LNA nucleotides. The oligonucleotide maycomprise alternating LNA nucleotides and 2′-O-methyl nucleotides. Theoligonucleotide may have a 5′ nucleotide that is a bridged nucleotide(e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). Theoligonucleotide may have a 5′ nucleotide that is a deoxyribonucleotide.

The oligonucleotide may comprise deoxyribonucleotides flanked by atleast one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide,ENA nucleotide) on each of the 5′ and 3′ ends of thedeoxyribonucleotides. The oligonucleotide may comprisedeoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridgednucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) oneach of the 5′ and 3′ ends of the deoxyribonucleotides. The 3′ positionof the oligonucleotide may have a 3′ hydroxyl group. The 3′ position ofthe oligonucleotide may have a 3′ thiophosphate.

The oligonucleotide may be conjugated with a label. For example, theoligonucleotide may be conjugated with a biotin moiety, cholesterol,Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such asCPP, hydrophobic molecules, such as lipids, ASGPR or dynamicpolyconjugates and variants thereof at its 5′ or 3′ end.

Preferably the single stranded oligonucleotide comprises one or moremodifications comprising: a modified sugar moiety, and/or a modifiedinternucleoside linkage, and/or a modified nucleotide and/orcombinations thereof. It is not necessary for all positions in a givenoligonucleotide to be uniformly modified, and in fact more than one ofthe modifications described herein may be incorporated in a singleoligonucleotide or even at within a single nucleoside within anoligonucleotide.

In some embodiments, the single stranded oligonucleotides are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric singlestranded oligonucleotides of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the single stranded oligonucleotide comprises atleast one nucleotide modified at the 2′ position of the sugar, mostpreferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modifiednucleotide. In other preferred embodiments, RNA modifications include2′-fluoro, 2′-amino and 2′ O-methyl modifications on the ribose ofpyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than 2′-deoxyoligonucleotides against agiven target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide; thesemodified oligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone, CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)— CH₂—CH₂ backbones, wherein thenative phosphodiester backbone is represented as O—P—O—CH,); amidebackbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374);morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991. In some embodiments, the morpholino-basedoligomeric compound is a phosphorodiamidate morpholino oligomer (PMO)(e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001;and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures ofwhich are incorporated herein by reference in their entireties).

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

Modified oligonucleotides are also known that include oligonucleotidesthat are based on or constructed from arabinonucleotide or modifiedarabinonucleotide residues. Arabinonucleosides are stereoisomers ofribonucleosides, differing only in the configuration at the 2′-positionof the sugar ring. In some embodiments, a 2′-arabino modification is2′-F arabino. In some embodiments, the modified oligonucleotide is2′-fluoro-D-arabinonucleic acid (FANA) (as described in, for example,Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med.Chem. Lett., 12:2651-2654, 2002; the disclosures of which areincorporated herein by reference in their entireties). Similarmodifications can also be made at other positions on the sugar,particularly the 3′ position of the sugar on a 3′ terminal nucleoside orin 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide. PCT Publication No. WO 99/67378 discloses arabinonucleicacids (ANA) oligomers and their analogues for improved sequence specificinhibition of gene expression via association to complementary messengerRNA.

Other preferred modifications include ethylene-bridged nucleic acids(ENAs) (e.g., International Patent Publication No. WO 2005/042777,Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al.,Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther.,8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf),49:171-172, 2005; the disclosures of which are incorporated herein byreference in their entireties). Preferred ENAs include, but are notlimited to, 2′-O,4′-C-ethylene-bridged nucleic acids.

Examples of LNAs are described in WO/2008/043753 and include compoundsof the following general formula.

where X and Y are independently selected among the groups —O—,

—S—, —N(H)—, N(R)—, —CH₂— or —CH— (if part of a double bond),

—CH₂—O—, —CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if partof a double bond),

—CH═CH—, where R is selected from hydrogen and C₁₋₄-alkyl; Z and Z* areindependently selected among an internucleoside linkage, a terminalgroup or a protecting group; B constitutes a natural or non-naturalnucleotide base moiety; and the asymmetric groups may be found in eitherorientation.

Preferably, the LNA used in the oligonucleotides described hereincomprises at least one LNA unit according any of the formulas

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleoside linkage, a terminal group or aprotecting group; B constitutes a natural or non-natural nucleotide basemoiety, and RH is selected from hydrogen and C₁₋₄-alkyl.

In some embodiments, the Locked Nucleic Acid (LNA) used in theoligonucleotides described herein comprises at least one Locked NucleicAcid (LNA) unit according any of the formulas shown in Scheme 2 ofPCT/DK2006/000512.

In some embodiments, the LNA used in the oligomer of the inventioncomprises internucleoside linkages selected from —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—,—O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected from hydrogen and C₁₋₄-alkyl.

Specifically preferred LNA units are shown in scheme 2:

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from S or —CH₂—S—.Thio-LNA can be in both beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from —N(H)—, N(R)—,CH₂—N(H)—, and —CH₂—N(R)— where R is selected from hydrogen andC₁₋₄-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above represents —O— or —CH₂—O—.Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is —CH₂—O— (where the oxygen atom of —CH₂—O— isattached to the 2′-position relative to the base B).

LNAs are described in additional detail herein.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)](Martin et al, Helv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2′-methoxy (2′-0-CH₃), 2′-propoxy(2′-OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Single stranded oligonucleotides can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, aswell as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,5-propynyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,2-chloro-6-aminopurine and 2,6-diaminopurine or other diaminopurines.See, e.g., Kornberg, “DNA Replication,” W. H. Freeman & Co., SanFrancisco, 1980, pp 75-77; and Gebeyehu, G., et al. Nucl. Acids Res.,15:4513 (1987)). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, in Crooke, andLebleu, eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and may be used as base substitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the modificationsdescribed herein may be incorporated in a single oligonucleotide or evenat within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science, 1991, 254, 1497-1500.

Single stranded oligonucleotides can also include one or more nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasescomprise the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) and uracil (U). Modifiednucleobases comprise other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in “The Concise Encyclopedia of PolymerScience And Engineering”, pages 858-859, Kroschwitz, ed. John Wiley &Sons, 1990; those disclosed by Englisch et al., Angewandle Chemie,International Edition, 1991, 30, page 613, and those disclosed bySanghvi. Chapter 15, Antisense Research and Applications,” pages289-302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi. etal., eds, “Antisense Research and Applications,” CRC Press, Boca Raton,1993, pp. 276-278) and are presently preferred base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications. Modified to nucleobases are described in U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617;5,750,692, and 5,681,941, each of which is herein incorporated byreference.

In some embodiments, the single stranded oligonucleotides are chemicallylinked to one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Forexample, one or more single stranded oligonucleotides, of the same ordifferent types, can be conjugated to each other; or single strandedoligonucleotides can be conjugated to targeting moieties with enhancedspecificity for a cell type or tissue type. Such moieties include, butare not limited to, lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al,Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996,277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941, each of which is herein incorporated byreference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

In some embodiments, single stranded oligonucleotide modificationinclude modification of the 5′ or 3′ end of the oligonucleotide. In someembodiments, the 3′ end of the oligonucleotide comprises a hydroxylgroup or a thiophosphate. It should be appreciated that additionalmolecules (e.g. a biotin moiety or a fluorophor) can be conjugated tothe 5′ or 3′ end of the single stranded oligonucleotide. In someembodiments, the single stranded oligonucleotide comprises a biotinmoiety conjugated to the 5′ nucleotide.

In some embodiments, the single stranded oligonucleotide compriseslocked nucleic acids (LNA), ENA modified nucleotides, 2′-O-methylnucleotides, or 2′-fluoro-deoxyribonucleotides. In some embodiments, thesingle stranded oligonucleotide comprises alternatingdeoxyribonucleotides and 2′-fluoro-deoxyribonucleotides. In someembodiments, the single stranded oligonucleotide comprises alternatingdeoxyribonucleotides and 2′-O-methyl nucleotides. In some embodiments,the single stranded oligonucleotide comprises alternatingdeoxyribonucleotides and ENA modified nucleotides. In some embodiments,the single stranded oligonucleotide comprises alternatingdeoxyribonucleotides and locked nucleic acid nucleotides. In someembodiments, the single stranded oligonucleotide comprises alternatinglocked nucleic acid nucleotides and 2′-O-methyl nucleotides.

In some embodiments, the 5′ nucleotide of the oligonucleotide is adeoxyribonucleotide. In some embodiments, the 5′ nucleotide of theoligonucleotide is a locked nucleic acid nucleotide. In someembodiments, the nucleotides of the oligonucleotide comprisedeoxyribonucleotides flanked by at least one locked nucleic acidnucleotide on each of the 5′ and 3′ ends of the deoxyribonucleotides. Insome embodiments, the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group or a 3′ thiophosphate.

In some embodiments, the single stranded oligonucleotide comprisesphosphorothioate internucleotide linkages. In some embodiments, thesingle stranded oligonucleotide comprises phosphorothioateinternucleotide linkages between at least two nucleotides. In someembodiments, the single stranded oligonucleotide comprisesphosphorothioate internucleotide linkages between all nucleotides.

It should be appreciated that the single stranded oligonucleotide canhave any combination of modifications as described herein.

The oligonucleotide may comprise a nucleotide sequence having one ormore of the following modification patterns.

(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,

(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx,(X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX,(X)xxxXXx, (X)xxxXxX and (X)xxxxXX,

(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx,(X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,

(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx, (X)XxxXXXX,(X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx,(X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,

(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx,and

(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, inwhich “X” denotes a nucleotide analogue, (X) denotes an optionalnucleotide analogue, and “x” denotes a DNA or RNA nucleotide unit. Eachof the above listed patterns may appear one or more times within anoligonucleotide, alone or in combination with any of the other disclosedmodification patterns.

Methods for Modulating Gene Expression

In some embodiments, methods are provided for increasing expression ofSMN protein in a cell. The methods, in some embodiments, involvedelivering to the cell a first single stranded oligonucleotidecomplementary with a PRC2-associated region of SMN1 or SMN2 and a secondsingle stranded oligonucleotide complementary with a splice controlsequence of a precursor mRNA of SMN1 or SMN2, in amounts sufficient toincrease expression of a mature mRNA of SMN1 or SMN2 that comprises (orincludes) exon 7 in the cell. The first and second single strandedoligonucleotides may be delivered together or separately. The first andsecond single stranded oligonucleotides may be linked together, orunlinked, i.e., separate.

In some embodiments, methods are provided for treating spinal muscularatrophy in a subject. The methods, in some embodiments, involveadministering to a subject a first single stranded oligonucleotidecomplementary with a PRC2-associated region of SMN1 or SMN2 and a secondsingle stranded oligonucleotide complementary with a splice controlsequence of a precursor mRNA of SMN1 or SMN2, in amounts sufficient toincrease expression of full length SMN protein in the subject to levelssufficient to improve one or more conditions associated with SMA. Thefirst and second single stranded oligonucleotides may be administeredtogether or separately. The first and second single strandedoligonucleotides may be linked together, or unlinked, i.e., separate.The first single stranded oligonucleotide may be administered within 1hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hoursor more of administration of the second single stranded oligonucleotide.The first single stranded oligonucleotide may be administered before orafter the second single stranded oligonucleotide. The oligonucleotidesmay be administered once or on multiple occasions depending on the needsof the subject and/or judgment of the treating physician. In some cases,the oligonucleotides may be administered in cycles. The administrationcycles may vary; for example, the administration cycle may be 2^(nd)oligo-1^(st) oligo-2^(nd) oligo-1^(st) oligo and so on; or 1^(st)oligo-2^(nd) oligo-1^(st) oligo-2^(nd) oligo, and so on; or 1^(st)oligo-2^(nd) oligo-2^(nd) oligo-1^(st) oligo-1^(st) oligo-2^(nd)oligo-2^(nd) oligo-1^(st) oligo, and so on. The skilled artisan will becapable of selecting administration cycles and intervals between eachadministration that are appropriate for treating a particular subject.

In one aspect, the invention relates to methods for modulating geneexpression in a cell (e.g., a cell for which SMN1 or SMN2 levels arereduced) for research purposes (e.g., to study the function of the genein the cell). In another aspect, the invention relates to methods formodulating gene expression in a cell (e.g., a cell for which SMN1 orSMN2 levels are reduced) for gene or epigenetic therapy. The cells canbe in vitro, ex vivo, or in vivo (e.g., in a subject who has a diseaseresulting from reduced expression or activity of SMN1 or SMN2. In someembodiments, methods for modulating gene expression in a cell comprisedelivering a single stranded oligonucleotide as described herein. Insome embodiments, delivery of the single stranded oligonucleotide to thecell results in a level of expression of gene that is at least 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater thana level of expression of gene in a control cell to which the singlestranded oligonucleotide has not been delivered. In certain embodiments,delivery of the single stranded oligonucleotide to the cell results in alevel of expression of gene that is at least 50% greater than a level ofexpression of gene in a control cell to which the single strandedoligonucleotide has not been delivered.

In another aspect of the invention, methods comprise administering to asubject (e.g. a human) a composition comprising a single strandedoligonucleotide as described herein to increase protein levels in thesubject. In some embodiments, the increase in protein levels is at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more,higher than the amount of a protein in the subject before administering.

As another example, to increase expression of SMN1 or SMN2 in a cell,the methods include introducing into the cell a single strandedoligonucleotide that is sufficiently complementary to a PRC2-associatedregion (e.g., of a long non-coding RNA) that maps to a genomic positionencompassing or in proximity to the SMN1 or SMN2 gene.

In another aspect of the invention provides methods of treating acondition (e.g., Spinal muscular atrophy) associated with decreasedlevels of expression of SMN1 or SMN2 in a subject, the method comprisingadministering a single stranded oligonucleotide as described herein.

A subject can include a non-human mammal, e.g. mouse, rat, guinea pig,rabbit, cat, dog, goat, cow, or horse. In preferred embodiments, asubject is a human. Single stranded oligonucleotides have been employedas therapeutic moieties in the treatment of disease states in animals,including humans. Single stranded oligonucleotides can be usefultherapeutic modalities that can be configured to be useful in treatmentregimes for the treatment of cells, tissues and animals, especiallyhumans.

For therapeutics, an animal, preferably a human, suspected of havingSpinal muscular atrophy is treated by administering single strandedoligonucleotide in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a single stranded oligonucleotide as described herein.

Formulation, Delivery, and Dosing

The oligonucleotides described herein can be formulated foradministration to a subject for treating a condition (e.g., Spinalmuscular atrophy) associated with decreased levels of SMN protein. Itshould be understood that the formulations, compositions and methods canbe practiced with any of the oligonucleotides disclosed herein. In someembodiments, formulations are provided that comprise a first singlestranded oligonucleotide complementary with a PRC2-associated region ofa gene and a second single stranded oligonucleotide complementary to asplice control sequence of a precursor mRNA of the gene. In someembodiments, formulations are provided that comprise a first singlestranded oligonucleotide complementary with a PRC2-associated region ofa gene that is linked via a linker with a second single strandedoligonucleotide complementary to a splice control sequence of aprecursor mRNA of the gene. Thus, it should be appreciated that in someembodiments, a first single stranded oligonucleotide complementary witha PRC2-associated region of a gene is linked with a second singlestranded oligonucleotide complementary to a splice control sequence of aprecursor mRNA of the gene, and in other embodiments, the singlestranded oligonucleotides are not linked. Single strandedoligonucleotides that are not linked may be administered to a subject ordelivered to a cell simultaneously (e.g., within the same composition)or separately.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient (e.g., an oligonucleotide or compound of theinvention) which can be combined with a carrier material to produce asingle dosage form will vary depending upon the host being treated, theparticular mode of administration, e.g., intradermal or inhalation. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect, e.g. tumorregression.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such formulations can contain sweetening agents, flavoring agents,coloring agents and preserving agents. A formulation can be admixturedwith nontoxic pharmaceutically acceptable excipients which are suitablefor manufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

A formulated single stranded oligonucleotide composition can assume avariety of states. In some examples, the composition is at leastpartially crystalline, uniformly crystalline, and/or anhydrous (e.g.,less than 80, 50, 30, 20, or 10% water). In another example, the singlestranded oligonucleotide is in an aqueous phase, e.g., in a solutionthat includes water. The aqueous phase or the crystalline compositionscan, e.g., be incorporated into a delivery vehicle, e.g., a liposome(particularly for the aqueous phase) or a particle (e.g., amicroparticle as can be appropriate for a crystalline composition).Generally, the single stranded oligonucleotide composition is formulatedin a manner that is compatible with the intended method ofadministration.

In some embodiments, the composition is prepared by at least one of thefollowing methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A single stranded oligonucleotide preparation can be formulated oradministered (together or separately) in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a singlestranded oligonucleotide, e.g., a protein that complexes with singlestranded oligonucleotide. Still other agents include chelators, e.g.,EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAseinhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin)and so forth. In some embodiments, the other agent used in combinationwith the single stranded oligonucleotide is an agent that also regulatesSMN expression. In some embodiments, the other agent is a growthhormone, a histone deacetylase inhibitor, a hydroxycarbamide(hydroxyurea), a natural polyphenol compound (e.g., resveratrol,curcumin), prolactin, or salbutamol. Examples of histone deacetylaseinhibitors that may be used include aliphatic compounds (e.g., butyrates(e.g., sodium butyrate and sodium phenylbutyrate) and valproic acid),benzamides (e.g., M344), and hydroxamic acids (e.g., CBHA, SBHA,Entinostat (MS-275)) Panobinostat (LBH-589), Trichostatin A, Vorinostat(SAHA)),

In one embodiment, the single stranded oligonucleotide preparationincludes another single stranded oligonucleotide, e.g., a second singlestranded oligonucleotide that modulates expression and/or mRNAprocessing of a second gene or a second single stranded oligonucleotidethat modulates expression of the first gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent single stranded oligonucleotide species. Such single strandedoligonucleotides can mediated gene expression with respect to a similarnumber of different genes. In one embodiment, the single strandedoligonucleotide preparation includes at least a second therapeutic agent(e.g., an agent other than an oligonucleotide).

Route of Delivery

A composition that includes a single stranded oligonucleotide can bedelivered to a subject by a variety of routes. Exemplary routes include:intravenous, intradermal, topical, rectal, parenteral, anal,intravaginal, intranasal, pulmonary, ocular. The term “therapeuticallyeffective amount” is the amount of oligonucleotide present in thecomposition that is needed to provide the desired level of SMN1 or SMN2expression in the subject to be treated to give the anticipatedphysiological response. The term “physiologically effective amount” isthat amount delivered to a subject to give the desired palliative orcurative effect. The term “pharmaceutically acceptable carrier” meansthat the carrier can be administered to a subject with no significantadverse toxicological effects to the subject.

The single stranded oligonucleotide molecules of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically include one or more speciesof single stranded oligonucleotide and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the single stranded oligonucleotide in aerosolform. The vascular endothelial cells could be targeted by coating aballoon catheter with the single stranded oligonucleotide andmechanically introducing the oligonucleotide.

Topical administration refers to the delivery to a subject by contactingthe formulation directly to a surface of the subject. The most commonform of topical delivery is to the skin, but a composition disclosedherein can also be directly applied to other surfaces of the body, e.g.,to the eye, a mucous membrane, to surfaces of a body cavity or to aninternal surface. As mentioned above, the most common topical deliveryis to the skin. The term encompasses several routes of administrationincluding, but not limited to, topical and transdermal. These modes ofadministration typically include penetration of the skin's permeabilitybarrier and efficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.Topical administration can also be used as a means to selectivelydeliver oligonucleotides to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy. In addition, iontophoresis (transferof ionic solutes through biological membranes under the influence of anelectric field), phonophoresis or sonophoresis (use of ultrasound toenhance the absorption of various therapeutic agents across biologicalmembranes, notably the skin and the cornea), and optimization of vehiclecharacteristics relative to dose position and retention at the site ofadministration may be useful methods for enhancing the transport oftopically applied compositions across skin and mucosal sites.

Both the oral and nasal membranes offer advantages over other routes ofadministration. For example, oligonucleotides administered through thesemembranes may have a rapid onset of action, provide therapeutic plasmalevels, avoid first pass effect of hepatic metabolism, and avoidexposure of the oligonucleotides to the hostile gastrointestinal (GI)environment. Additional advantages include easy access to the membranesites so that the oligonucleotide can be applied, localized and removedeasily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many agents. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

A pharmaceutical composition of single stranded oligonucleotide may alsobe administered to the buccal cavity of a human being by spraying intothe cavity, without inhalation, from a metered dose spray dispenser, amixed micellar pharmaceutical formulation as described above and apropellant. In one embodiment, the dispenser is first shaken prior tospraying the pharmaceutical formulation and propellant into the buccalcavity.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, slurries, emulsions, elixirsor non-aqueous media, tablets, capsules, lozenges, or troches. In thecase of tablets, carriers that can be used include lactose, sodiumcitrate and salts of phosphoric acid. Various disintegrants such asstarch, and lubricating agents such as magnesium stearate, sodium laurylsulfate and talc, are commonly used in tablets. For oral administrationin capsule form, useful diluents are lactose and high molecular weightpolyethylene glycols. When aqueous suspensions are required for oraluse, the nucleic acid compositions can be combined with emulsifying andsuspending agents. If desired, certain sweetening and/or flavoringagents can be added.

Parenteral administration includes intravenous drip, subcutaneous,intraperitoneal or intramuscular injection, intrathecal orintraventricular administration. In some embodiments, parentaladministration involves administration directly to the site of disease(e.g. injection into a tumor).

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

Any of the single stranded oligonucleotides described herein can beadministered to ocular tissue. For example, the compositions can beapplied to the surface of the eye or nearby tissue, e.g., the inside ofthe eyelid. For ocular administration, ointments or droppable liquidsmay be delivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers. The single stranded oligonucleotide can alsobe administered to the interior of the eye, and can be introduced by aneedle or other delivery device which can introduce it to a selectedarea or structure.

Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably singlestranded oligonucleotides, within the dispersion can reach the lungwhere it can be readily absorbed through the alveolar region directlyinto blood circulation. Pulmonary delivery can be effective both forsystemic delivery and for localized delivery to treat diseases of thelungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver agents that may be readily formulated as dry powders. A singlestranded oligonucleotide composition may be stably stored as lyophilizedor spray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred. Pulmonary administration of amicellar single stranded oligonucleotide formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Exemplary devices include devices which are introduced into thevasculature, e.g., devices inserted into the lumen of a vascular tissue,or which devices themselves form a part of the vasculature, includingstents, catheters, heart valves, and other vascular devices. Thesedevices, e.g., catheters or stents, can be placed in the vasculature ofthe lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to asingle stranded oligonucleotide, e.g., a device can release insulin.

In one embodiment, unit doses or measured doses of a composition thatincludes single stranded oligonucleotide are dispensed by an implanteddevice. The device can include a sensor that monitors a parameter withina subject. For example, the device can include pump, e.g., and,optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with a single strandedoligonucleotide, ex vivo and then administered or implanted in asubject. The tissue can be autologous, allogeneic, or xenogeneic tissue.E.g., tissue can be treated to reduce graft v. host disease. In otherembodiments, the tissue is allogeneic and the tissue is treated to treata disorder characterized by unwanted gene expression in that tissue.E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoieticcells, can be treated to inhibit unwanted cell proliferation.Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies. In some implementations, the singlestranded oligonucleotide treated cells are insulated from other cells,e.g., by a semi-permeable porous barrier that prevents the cells fromleaving the implant, but enables molecules from the body to reach thecells and molecules produced by the cells to enter the body. In oneembodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains asingle stranded oligonucleotide. Exemplary devices include condoms,diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths,and birth control devices.

Dosage

In one aspect, the invention features a method of administering a singlestranded oligonucleotide (e.g., as a compound or as a component of acomposition) to a subject (e.g., a human subject). In some embodiments,the methods involve administering a compound (e.g., a compound of thegeneral formula A-B-C, as disclosed herein, or an single strandedoligonucleotide,) in a unit dose to a subject. In one embodiment, theunit dose is between about 10 mg and 25 mg per kg of bodyweight. In oneembodiment, the unit dose is between about 1 mg and 100 mg per kg ofbodyweight. In one embodiment, the unit dose is between about 0.1 mg and500 mg per kg of bodyweight. In some embodiments, the unit dose is morethan 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mgper kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with theSMN1 or SMN2. The unit dose, for example, can be administered byinjection (e.g., intravenous or intramuscular), an inhaled dose, or atopical application.

In some embodiments, the unit dose is administered daily. In someembodiments, less frequently than once a day, e.g., less than every 2,4, 8 or 30 days. In another embodiment, the unit dose is notadministered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. In someembodiments, the unit dose is administered more than once a day, e.g.,once an hour, two hours, four hours, eight hours, twelve hours, etc.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a single stranded oligonucleotide. Themaintenance dose or doses are generally lower than the initial dose,e.g., one-half less of the initial dose. A maintenance regimen caninclude treating the subject with a dose or doses ranging from 0.0001 to100 mg/kg of body weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or0.0001 mg per kg of bodyweight per day. The maintenance doses may beadministered no more than once every 1, 5, 10, or 30 days. Further, thetreatment regimen may last for a period of time which will varydepending upon the nature of the particular disease, its severity andthe overall condition of the patient. In some embodiments the dosage maybe delivered no more than once per day, e.g., no more than once per 24,36, 48, or more hours, e.g., no more than once for every 5 or 8 days.Following treatment, the patient can be monitored for changes in hiscondition and for alleviation of the symptoms of the disease state. Thedosage of the oligonucleotide may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In some embodiments, the pharmaceutical composition includes a pluralityof single stranded oligonucleotide species. In some embodiments, thepharmaceutical composition comprises a first single strandedoligonucleotide complementary with a PRC2-associated region of a gene(e.g., SMN1 or SMN2), and a second single stranded oligonucleotidecomplementary to a splice control sequence of a precursor mRNA of a gene(e.g., SMN1 or SMN2). In some embodiments, the pharmaceuticalcomposition includes a compound comprising the general formula A-B-C, inwhich A is a single stranded oligonucleotide complementary with aPRC2-associated region of a gene, B is a linker, and C is a singlestranded oligonucleotide complementary to a splice control sequence of aprecursor mRNA of the gene.

In another embodiment, the single stranded oligonucleotide species hassequences that are non-overlapping and non-adjacent to another specieswith respect to a naturally occurring target sequence (e.g., aPRC2-associated region). In another embodiment, the plurality of singlestranded oligonucleotide specics is specific for differentPRC2-associated regions. In another embodiment, the single strandedoligonucleotide is allele specific. In some cases, a patient is treatedwith a single stranded oligonucleotide in conjunction with othertherapeutic modalities.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.0001 mg to 100 mg per kg of bodyweight.

The concentration of the single stranded oligonucleotide composition isan amount sufficient to be effective in treating or preventing adisorder or to regulate a physiological condition in humans. Theconcentration or amount of single stranded oligonucleotide administeredwill depend on the parameters determined for the agent and the method ofadministration, e.g. nasal, buccal, pulmonary. For example, nasalformulations may tend to require much lower concentrations of someingredients in order to avoid irritation or burning of the nasalpassages. It is sometimes desirable to dilute an oral formulation up to10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a single strandedoligonucleotide can include a single treatment or, preferably, caninclude a series of treatments. It will also be appreciated that theeffective dosage of a single stranded oligonucleotide used for treatmentmay increase or decrease over the course of a particular treatment. Forexample, the subject can be monitored after administering a singlestranded oligonucleotide composition. Based on information from themonitoring, an additional amount of the single stranded oligonucleotidecomposition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of SMN1 or SMN2 expression levels in thebody of the patient. Persons of ordinary skill can easily determineoptimum dosages, dosing methodologies and repetition rates. Optimumdosages may vary depending on the relative potency of individualcompounds, and can generally be estimated based on EC50s found to beeffective in in vitro and in vivo animal models. In some embodiments,the animal models include transgenic animals that express a human SMN1or SMN2. In another embodiment, the composition for testing includes asingle stranded oligonucleotide that is complementary, at least in aninternal region, to a sequence that is conserved between SMN1 or SMN2 inthe animal model and the SMN1 or SMN2 in a human.

In one embodiment, the administration of the single strandedoligonucleotide composition is parenteral, e.g. intravenous (e.g., as abolus or as a diffusible infusion), intradermal, intraperitoneal,intramuscular, intrathecal, intraventricular, intracranial,subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal,oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.Administration can be provided by the subject or by another person,e.g., a health care provider. The composition can be provided inmeasured doses or in a dispenser which delivers a metered dose. Selectedmodes of delivery are discussed in more detail below.

Kits

In certain aspects of the invention, kits are provided, comprising acontainer housing a composition comprising a single strandedoligonucleotide. In some embodiments, the kits comprise a containerhousing a single stranded oligonucleotide complementary with of aPRC2-associated region of a gene; and a second container housing asingle stranded oligonucleotide complementary to a splice controlsequence of a precursor mRNA of the gene. In some embodiments, the kitscomprise a container housing a single stranded oligonucleotidecomplementary with of a PRC2-associated region of a gene and a singlestranded oligonucleotide complementary to a splice control sequence of aprecursor mRNA of the gene. In some embodiments, the composition is apharmaceutical composition comprising a single stranded oligonucleotideand a pharmaceutically acceptable carrier. In some embodiments, theindividual components of the pharmaceutical composition may be providedin one container. Alternatively, it may be desirable to provide thecomponents of the pharmaceutical composition separately in two or morecontainers, e.g., one container for single stranded oligonucleotides,and at least another for a carrier compound. The kit may be packaged ina number of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

INCORPORATION BY REFERENCE

International Patent Appln. Pub. No. WO 2013/173638, entitledCOMPOSITIONS AND METHODS FOR MODULATING SMN GENE FAMILY EXPRESSION,International Patent Appln. Pub. No. WO 2013/173645, entitledCOMPOSITIONS AND METHODS FOR MODULATING UTRN EXPRESSION, InternationalPatent Appln. Pub. No. WO 2013/173599, entitled COMPOSITIONS AND METHODSFOR MODULATING HEMOGLOBIN GENE FAMILY EXPRESSION, International PatentAppln. Pub. No. WO 2013/173598, entitled COMPOSITIONS AND METHODS FORMODULATING ATP2A2 EXPRESSION, International Patent Appln. Pub. No. WO2013/173647, entitled to COMPOSITIONS AND METHODS FOR MODULATING APOA1AND ABCA1 EXPRESSION, International Patent Appln. Pub. No. WO2013/173605, entitled COMPOSITIONS AND METHODS FOR MODULATING PTENEXPRESSION, International Patent Appln. Pub. No. WO 2013/173601,entitled COMPOSITIONS AND METHODS FOR MODULATING BDNF EXPRESSION,International Patent Appln. Pub. No. WO 2013/173608, entitledCOMPOSITIONS AND METHODS FOR MODULATING MECP2 EXPRESSION, InternationalPatent Appln. Pub. No. WO 2013/173635, entitled COMPOSITIONS AND METHODSFOR MODULATING GENE EXPRESSION, International Patent Appln. Pub. No. WO2013/173637, entitled COMPOSITIONS AND METHODS FOR MODULATING GENEEXPRESSION, and International Patent Appln. Pub. No. WO 2013/173652,entitled COMPOSITIONS AND METHODS FOR MODULATING GENE EXPRESSION areincorporated herein by reference in their entirety.

Appendix A contains Table 5. Appendix A can be found on pages 106-287 ofWO 2013/173638, which are incorporated by reference herein in theirentirety. Appendix B contains Table 6. Appendix B can be found on pages288-309 of WO 2013/173638, which are incorporated by reference herein intheir entirety. Appendix C contains Table 7. Appendix C can be found onpages 310-399 of WO 2013/173638, which are incorporated by referenceherein in their entirety.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1: Oligonucleotides Targeting PRC2-Associated Regions thatUpregulate SMN1

Materials and Methods:

Real Time PCR

RNA was harvested from the cells using Promega SV 96 Total RNA Isolationsystem or Trizol omitting the DNAse step. In separate pilot experiments,50 ng of RNA was determined to be sufficient template for the reversetranscriptase reaction. RNA harvested from cells was normalized so that50 ng of RNA was input to each reverse transcription reaction. For thefew samples that were too dilute to reach this limit, the maximum inputvolume was added. Reverse transcriptase reaction was performed using theSuperscript II kit and real time PCR performed on cDNA samples usingicycler SYBR green chemistry (Biorad). A baseline level of mRNAexpression for each target gene was determined through quantitative PCRas outlined above. Baseline levels were also determined for mRNA ofvarious housekeeping genes which are constitutively expressed. A“control” housekeeping gene with approximately the same level ofbaseline expression as the target gene was chosen for comparisonpurposes.

Protein Expression (ELISA)

ELISA to determine SMN protein was carried out per manufacturer'sinstructions (SMN ELISA kit # ADI-900-209, Enzo Life Sciences).

Cell Culture

Human hepatocyte Hep3B, human hepatocyte HepG2 cells, mouse hepatomaHepa1-6 cells, and human renal proximal tubule epithelial cells (RPTEC)were cultured using conditions known in the art (see, e.g. CurrentProtocols in Cell Biology). Other cell lines tested were neuronal celllines (SK-N-AS, U-87) and SMN patient fibroblasts. Details of the celllines used in the experiments described herein are provided in Table 8.

TABLE 8 Cell lines Culture Cell line Source Species Gender Cell TypeTissue Status Conditions RPTEC Lonza human N/A proximal kidney primaryClonetics ™ tubule REGM ™ epithelial BulletKit ™ cells (CC-3190) Hep3BATCC human M hepatocytes liver immortalized Eagle's MEM + 10% FBSSK-N-AS ATCC human F neuroblast brain immortalized DMEM + 10% FBS U-87ATCC human M gliobastoma brain immortalized Eagle's MEM + 10% FBSGM03813 Coriell human F fibroblast skin immortalized MEM + 10% FBSInstitute GM03814 Coriell human M fibroblast skin immortalized MEM + 10%FBS Institute GM09677 Coriell human M fibroblast skin immortalized MEM +10% FBS Institute GM00232 Coriell human M fibroblast skin immortalizedMEM + 10% FBS Institute GM03815 Coriell human M fibroblast skinimmortalized MEM + 10% FBS Institute GM22592 Coriell human M fibroblastskin immortalized MEM + 10% FBS Institute GM10684 Coriell human FB-lymphocyte blood immortalized MEM + 10% FBS Institute GM00321 Coriellhuman F fibroblast skin immortalized MEM + 10% FBS (normal) InstituteOligonucleotide Design

Oligonucleotides were designed within PRC2-interacting regions in orderto upregulate SMN1. The sequence and structure of each oligonucleotideis shown in Table 2. The following table provides a description of thenucleotide analogs, modifications and intranucleotide linkages used forcertain oligonucleotides tested and described in Table 2.

In Vitro Transfection of Cells with Oligonucleotides

Cells were seeded into each well of 24-well plates at a density of25,000 cells per 500 uL and transfections were performed withLipofectamine and the single stranded oligonucleotides. Control wellscontained Lipofectamine alone. At 48 hours post-transfection,approximately 200 uL of cell culture supernatants were stored at −80 Cfor ELISA. At 48 hours post-transfection, RNA was harvested from thecells and quantitative PCR was carried out as outlined above. Thepercent induction of target mRNA expression by each oligonucleotide wasdetermined by normalizing mRNA levels in the presence of theoligonucleotide to the mRNA levels in the presence of control(Lipofectamine alone). This was compared side-by-side with the increasein mRNA expression of the “control” housekeeping gene.

Results:

In Vitro Delivery of Single Stranded Oligonucleotides Upregulated SMN1Expression

Oligonucleotides were designed as candidates for upregulating SMN1expression. A total of 52 single stranded oligonucleotides were designedto be complementary to a PRC2-interacting region within a sequence asset forth in SEQ ID NO: 1, 2, 4, or 5. Oligonucleotides were tested inat least duplicate. The sequence and structural features of theoligonucleotides are set forth in Table 2. Briefly, cells weretransfected in vitro with the oligonucleotides as described above. SMN1expression in cells following treatment was evaluated by qRT-PCR.Oligonucleotides that upregulated SMN1 expression were identified.Further details are outlined in Table 2.

Table 5 shows further results from experiments in which oligonucleotideswere transfected into cells at a particular concentration [oligo] and 48or 72 h later RNA was prepared and qRTPCR assays carried out todetermine mRNA levels of full length (FL) or delta7 SMN. In other cases,oligos were administered gymnotically to cells at 10 μM and RNAharvested 9 days post treatment. The cell lines tested were neuronalcell lines (SK-N-AS, U-87) and SMN patient fibroblasts.

Table 6 shows results from experiments in which oligonucleotides weretransfected into cells in combination with either one or two more oligosor small molecule compounds at a particular concentration ([oligo],[2nd], [3rd]) and 48 or 72 h later RNA was prepared and qRTPCR assayscarried out to determine mRNA levels of full length (FL) or delta7 SMN.The cell lines tested were SMN patient fibroblasts.

Table 7 shows results from experiments in which oligonucleotides weretransfected into cells in combination with either one or two more oligosor as dimers or by gymnotic treatment at a particular concentration([oligo], [2nd], [3rd]) and 24, 48, 72 or 216 h later cell lysates wereprepared and ELISA assays carried out to determine SMN protein levels.The cell lines tested were SMN patient fibroblasts.

TABLES

TABLE 1 Non-Seed hexamer sequences.AAAAAA, AAAAAG, AAAACA, AAAAGA, AAAAGC, AAAAGG, AAAAUA, AAACAA, AAACAC, AAACAG,AAACAU, AAACCC, AAACCU, AAACGA, AAACGC, AAACGU, AAACUA, AAACUC, AAACUU, AAAGAU,AAAGCC, AAAGGA, AAAGGG, AAAGUC, AAAUAC, AAAUAU, AAAUCG, AAAUCU, AAAUGC, AAAUGU,AAAUUA, AAAUUG, AACAAC, AACAAG, AACAAU, AACACA, AACACG, AACAGA, AACAGC, AACAGG,AACAUC, AACAUG, AACCAA, AACCAC, AACCAG, AACCAU, AACCCC, AACCCG, AACCGA, AACCGC,AACCGG, AACCUA, AACCUU, AACGAA, AACGAC, AACGAG, AACGAU, AACGCU, AACGGG, AACGGU,AACGUA, AACGUC, AACGUG, AACGUU, AACUAU, AACUCA, AACUCC, AACUCG, AACUGA, AACUGC,AACUGU, AACUUA, AACUUC, AACUUG, AACUUU, AAGAAA, AAGAAG, AAGAAU, AAGACG, AAGAGA,AAGAGC, AAGAGG, AAGAGU, AAGAUU, AAGCAA, AAGCAC, AAGCAG, AAGCAU, AAGCCA, AAGCCC,AAGCCG, AAGCCU, AAGCGA, AAGCGG, AAGCGU, AAGCUA, AAGGAA, AAGGAC, AAGGCU, AAGGGC,AAGGGU, AAGGUU, AAGUAA, AAGUAC, AAGUAU, AAGUCC, AAGUCG, AAGUGA, AAGUGG, AAGUUA,AAGUUU, AAUAAA, AAUAAC, AAUAAG, AAUAAU, AAUACA, AAUACC, AAUACG, AAUAGA, AAUAGC,AAUAGG, AAUAGU, AAUAUC, AAUAUU, AAUCAA, AAUCAU, AAUCCA, AAUCCC, AAUCCG, AAUCGA,AAUCGC, AAUCGU, AAUCUA, AAUCUG, AAUCUU, AAUGAA, AAUGAC, AAUGAG, AAUGAU, AAUGCG,AAUGCU, AAUGGA, AAUGGU, AAUGUA, AAUGUC, AAUGUG, AAUUAA, AAUUAC, AAUUAG, AAUUCC,AAUUCG, AAUUGA, AAUUGG, AAUUGU, AAUUUC, AAUUUG, ACAAAA, ACAAAC, ACAAAG, ACAAAU,ACAACC, ACAACG, ACAACU, ACAAGA, ACAAGC, ACAAGU, ACAAUC, ACAAUG, ACAAUU, ACACAG,ACACCA, ACACCC, ACACCG, ACACCU, ACACGA, ACACGC, ACACGU, ACACUC, ACACUG, ACACUU,ACAGAA, ACAGAC, ACAGCC, ACAGCG, ACAGCU, ACAGGG, ACAGUC, ACAGUG, ACAGUU, ACAUAA,ACAUAC, ACAUCC, ACAUCG, ACAUCU, ACAUGA, ACAUGC, ACAUGU, ACAUUG, ACAUUU, ACCAAA,ACCAAC, ACCAAG, ACCAAU, ACCACC, ACCACG, ACCAGA, ACCAGU, ACCAUA, ACCAUG, ACCAUU,ACCCAA, ACCCAC, ACCCCA, ACCCCG, ACCCGA, ACCCGC, ACCCUA, ACCCUC, ACCCUU, ACCGAA,ACCGAC, ACCGAU, ACCGCA, ACCGCC, ACCGCG, ACCGCU, ACCGGA, ACCGGC, ACCGGU, ACCGUA,ACCGUC, ACCGUG, ACCGUU, ACCUAA, ACCUAC, ACCUAG, ACCUAU, ACCUCA, ACCUCC, ACCUCG,ACCUCU, ACCUGA, ACCUGC, ACCUGU, ACCUUA, ACCUUC, ACCUUU, ACGAAA, ACGAAC, ACGAAG,ACGAAU, ACGACA, ACGACC, ACGACG, ACGACU, ACGAGA, ACGAGC, ACGAGG, ACGAGU, ACGAUA,ACGAUC, ACGAUG, ACGAUU, ACGCAA, ACGCAG, ACGCAU, ACGCCC, ACGCCG, ACGCCU, ACGCGA,ACGCGG, ACGCGU, ACGCUA, ACGCUG, ACGCUU, ACGGAA, ACGGAC, ACGGAG, ACGGAU, ACGGCC,ACGGCG, ACGGCU, ACGGGC, ACGGGG, ACGGGU, ACGGUA, ACGGUC, ACGGUG, ACGGUU, ACGUAA,ACGUAC, ACGUAU, ACGUCC, ACGUCG, ACGUCU, ACGUGA, ACGUGC, ACGUGG, ACGUGU, ACGUUA,ACGUUC, ACGUUG, ACGUUU, ACUAAA, ACUAAG, ACUAAU, ACUACA, ACUACC, ACUACG, ACUACU,ACUAGG, ACUAUC, ACUAUG, ACUAUU, ACUCAU, ACUCCC, ACUCCG, ACUCCU, ACUCGA, ACUCGC,ACUCGG, ACUCUC, ACUCUU, ACUGAG, ACUGAU, ACUGCC, ACUGCG, ACUGCU, ACUGGG, ACUGGU,ACUGUC, ACUUAA, ACUUAC, ACUUAU, ACUUCA, ACUUCC, ACUUCG, ACUUCU, ACUUGA, ACUUGC,ACUUGU, ACUUUA, ACUUUC, ACUUUG, AGAAAA, AGAAAC, AGAAAG, AGAACC, AGAACG, AGAACU,AGAAGC, AGAAGU, AGAAUA, AGAAUC, AGAAUG, AGAAUU, AGACAA, AGACAC, AGACAU, AGACCA,AGACCC, AGACCG, AGACCU, AGACGA, AGACGC, AGACGU, AGACUA, AGACUC, AGACUU, AGAGAC,AGAGAG, AGAGAU, AGAGCC, AGAGCG, AGAGCU, AGAGGC, AGAGGG, AGAGGU, AGAGUA, AGAGUU,AGAUAC, AGAUAG, AGAUAU, AGAUCC, AGAUCG, AGAUCU, AGAUGA, AGAUGC, AGAUGG, AGAUUA,AGAUUC, AGAUUG, AGAUUU, AGCAAC, AGCACA, AGCACG, AGCACU, AGCAGA, AGCAUA, AGCAUC,AGCAUG, AGCCAA, AGCCAU, AGCCCA, AGCCGA, AGCCGC, AGCCGG, AGCCGU, AGCCUA, AGCCUC,AGCGAA, AGCGAG, AGCGAU, AGCGCA, AGCGCC, AGCGCG, AGCGCU, AGCGGA, AGCGGC, AGCGGU,AGCGUA, AGCGUC, AGCGUG, AGCGUU, AGCUAA, AGCUAC, AGCUAG, AGCUAU, AGCUCA, AGCUCC,AGCUCG, AGCUCU, AGCUGA, AGCUGG, AGCUGU, AGCUUC, AGCUUU, AGGAAU, AGGACC, AGGACG,AGGAGA, AGGAGU, AGGAUA, AGGCAA, AGGCAU, AGGCCG, AGGCGA, AGGCGC, AGGCGG, AGGCUA,AGGCUC, AGGCUU, AGGGAC, AGGGAU, AGGGGA, AGGGGU, AGGGUA, AGGGUG, AGGUAA,AGGUAC, AGGUCA, AGGUCC, AGGUCU, AGGUGA, AGGUGC, AGGUGG, AGGUGU, AGGUUC,AGGUUG, AGUAAA, AGUAAG, AGUAAU, AGUACA, AGUACG, AGUAGC, AGUAGG, AGUAUA, AGUAUC,AGUAUG, AGUAUU, AGUCAA, AGUCAC, AGUCAG, AGUCAU, AGUCCA, AGUCCG, AGUCCU, AGUCGA,AGUCGC, AGUCGG, AGUCGU, AGUCUA, AGUCUC, AGUCUG, AGUCUU, AGUGAA, AGUGAC, AGUGCG,AGUGGG, AGUGUC, AGUUAA, AGUUAC, AGUUAG, AGUUCC, AGUUCG, AGUUGA, AGUUGC,AGUUGU, AGUUUA, AGUUUC, AGUUUG, AGUUUU, AUAAAC, AUAAAU, AUAACA, AUAACC, AUAACG,AUAACU, AUAAGA, AUAAGC, AUAAGG, AUAAGU, AUAAUC, AUAAUG, AUAAUU, AUACAC, AUACAG,AUACAU, AUACCA, AUACCC, AUACCG, AUACGA, AUACGC, AUACGG, AUACGU, AUACUA, AUACUC,AUACUG, AUACUU, AUAGAA, AUAGAC, AUAGAU, AUAGCA, AUAGCG, AUAGCU, AUAGGA, AUAGGU,AUAGUA, AUAGUC, AUAGUG, AUAGUU, AUAUAC, AUAUAG, AUAUCC, AUAUCG, AUAUCU, AUAUGA,AUAUGC, AUAUGG, AUAUGU, AUAUUC, AUAUUG, AUAUUU, AUCAAA, AUCAAC, AUCAAG, AUCAAU,AUCACA, AUCACC, AUCACG, AUCAGC, AUCAGG, AUCCAA, AUCCAU, AUCCCC, AUCCCG, AUCCGA,AUCCGC, AUCCGG, AUCCUA, AUCCUC, AUCCUG, AUCGAA, AUCGAC, AUCGAG, AUCGAU, AUCGCA,AUCGCC, AUCGCG, AUCGCU, AUCGGC, AUCGGG, AUCGGU, AUCGUC, AUCGUG, AUCGUU, AUCUAA,AUCUAC, AUCUAG, AUCUAU, AUCUCC, AUCUCG, AUCUGU, AUCUUG, AUCUUU, AUGAAA, AUGAAC,AUGAAG, AUGAAU, AUGACC, AUGACU, AUGAGG, AUGAGU, AUGAUA, AUGAUC, AUGAUU, AUGCAA,AUGCAG, AUGCCA, AUGCCC, AUGCCG, AUGCGA, AUGCGG, AUGCGU, AUGCUC, AUGCUU, AUGGAC,AUGGCC, AUGGGA, AUGGGC, AUGGGU, AUGGUC, AUGGUG, AUGUAC, AUGUAU, AUGUCA,AUGUCC, AUGUCG, AUGUGU, AUGUUA, AUGUUC, AUUAAA, AUUAAC, AUUAAG, AUUAAU, AUUACA,AUUACC, AUUACG, AUUACU, AUUAGA, AUUAGC, AUUAGG, AUUAGU, AUUAUA, AUUAUC, AUUAUG,AUUCAC, AUUCCA, AUUCCG, AUUCCU, AUUCGA, AUUCGC, AUUCGG, AUUCGU, AUUCUA, AUUCUC,AUUCUU, AUUGAA, AUUGAC, AUUGAU, AUUGCC, AUUGCG, AUUGCU, AUUGGA, AUUGGC,AUUGGG, AUUGGU, AUUGUA, AUUGUC, AUUGUG, AUUGUU, AUUUAA, AUUUAG, AUUUAU,AUUUCC, AUUUCG, AUUUCU, AUUUGA, AUUUGC, AUUUGU, AUUUUA, AUUUUC, AUUUUG,AUUUUU, CAAAAG, CAAACA, CAAACC, CAAACG, CAAACU, CAAAGA, CAAAGG, CAAAUA, CAAAUU,CAACAC, CAACAU, CAACCA, CAACCC, CAACCG, CAACGA, CAACGC, CAACGG, CAACGU, CAACUA,CAACUC, CAACUG, CAACUU, CAAGAA, CAAGAC, CAAGAU, CAAGCA, CAAGCC, CAAGCG, CAAGCU,CAAGGA, CAAGGG, CAAGUC, CAAGUG, CAAGUU, CAAUAA, CAAUAC, CAAUAG, CAAUCC, CAAUCG,CAAUCU, CAAUGA, CAAUGC, CAAUGG, CAAUGU, CAAUUC, CAAUUG, CAAUUU, CACAAU, CACACA,CACACG, CACACU, CACAGA, CACAGC, CACAGG, CACAUA, CACAUC, CACAUU, CACCAA, CACCAC,CACCAU, CACCCA, CACCCC, CACCCG, CACCGA, CACCGC, CACCGG, CACCGU, CACCUA, CACCUU,CACGAA, CACGAC, CACGAG, CACGAU, CACGCA, CACGCC, CACGCU, CACGGA, CACGGC, CACGGG,CACGGU, CACGUA, CACGUC, CACGUG, CACGUU, CACUAA, CACUAG, CACUAU, CACUCA, CACUCG,CACUGA, CACUGC, CACUGG, CACUUA, CACUUC, CACUUU, CAGAAA, CAGAAG, CAGAAU, CAGACC,CAGACG, CAGAGC, CAGAUA, CAGAUC, CAGCCG, CAGCCU, CAGCGA, CAGCGC, CAGCGG, CAGCGU,CAGCUC, CAGCUU, CAGGAU, CAGGGG, CAGGGU, CAGGUA, CAGGUC, CAGGUU, CAGUAC, CAGUCG,CAGUUG, CAUAAA, CAUAAC, CAUAAG, CAUAAU, CAUACA, CAUACC, CAUACG, CAUACU, CAUAGA,CAUAGG, CAUAGU, CAUAUA, CAUAUC, CAUAUG, CAUCAA, CAUCAC, CAUCAG, CAUCAU, CAUCCA,CAUCCC, CAUCCG, CAUCGA, CAUCGC, CAUCGG, CAUCGU, CAUCUA, CAUCUC, CAUCUG, CAUCUU,CAUGAA, CAUGAC, CAUGAG, CAUGAU, CAUGCA, CAUGCC, CAUGCG, CAUGCU, CAUGGC, CAUGGG,CAUGGU, CAUGUA, CAUGUC, CAUGUU, CAUUAA, CAUUAC, CAUUAG, CAUUCA, CAUUCC, CAUUCG,CAUUCU, CAUUGA, CAUUGG, CAUUUC, CAUUUG, CAUUUU, CCAAAA, CCAAAC, CCAAAG, CCAAAU,CCAACA, CCAACC, CCAACG, CCAACU, CCAAGA, CCAAGC, CCAAGG, CCAAUC, CCAAUG, CCAAUU,CCACAA, CCACAC, CCACAG, CCACAU, CCACCA, CCACCC, CCACCG, CCACCU, CCACGA, CCACGC,CCACGG, CCACGU, CCACUA, CCACUC, CCACUU, CCAGAA, CCAGAC, CCAGAG, CCAGCC, CCAGGU,CCAGUC, CCAGUU, CCAUAA, CCAUAC, CCAUAG, CCAUAU, CCAUCA, CCAUCC, CCAUCU, CCAUGA,CCAUGC, CCAUGG, CCAUUC, CCAUUG, CCAUUU, CCCAAC, CCCAAG, CCCAAU, CCCACA, CCCAGA,CCCAGC, CCCAGU, CCCAUA, CCCAUC, CCCAUG, CCCAUU, CCCCAA, CCCCAG, CCCCAU, CCCCCC,CCCCCG, CCCCCU, CCCCGA, CCCCGC, CCCCGU, CCCCUA, CCCCUC, CCCGAA, CCCGAC, CCCGAU,CCCGCA, CCCGCU, CCCGGA, CCCGGC, CCCGUA, CCCGUG, CCCGUU, CCCUAA, CCCUAG, CCCUCA,CCCUCU, CCCUGC, CCCUUA, CCCUUC, CCCUUU, CCGAAA, CCGAAC, CCGAAU, CCGACA, CCGACC,CCGACG, CCGACU, CCGAGA, CCGAGG, CCGAGU, CCGAUA, CCGAUC, CCGAUG, CCGAUU, CCGCAA,CCGCAC, CCGCAG, CCGCAU, CCGCCA, CCGCCC, CCGCCG, CCGCCU, CCGCGA, CCGCGC, CCGCGG,CCGCGU, CCGCUA, CCGCUC, CCGCUG, CCGCUU, CCGGAA, CCGGAU, CCGGCA, CCGGCC, CCGGCG,CCGGCU, CCGGGA, CCGGGC, CCGGGG, CCGGGU, CCGGUA, CCGGUC, CCGGUG, CCGUAA, CCGUAG,CCGUAU, CCGUCA, CCGUCC, CCGUCG, CCGUGA, CCGUGU, CCGUUA, CCGUUC, CCGUUG, CCGUUU,CCUAAC, CCUAAG, CCUAAU, CCUACA, CCUACC, CCUACG, CCUACU, CCUAGA, CCUAGC, CCUAGG,CCUAGU, CCUAUA, CCUAUC, CCUAUG, CCUAUU, CCUCAA, CCUCAC, CCUCAG, CCUCAU, CCUCCA,CCUCCC, CCUCCG, CCUCGA, CCUCGC, CCUCGG, CCUCGU, CCUCUA, CCUCUG, CCUGAC, CCUGAU,CCUGCA, CCUGGG, CCUGGU, CCUGUU, CCUUAA, CCUUAC, CCUUAG, CCUUAU, CCUUCG, CCUUGA,CCUUGU, CCUUUA, CCUUUC, CCUUUU, CGAAAA, CGAAAC, CGAAAG, CGAAAU, CGAACA, CGAACC,CGAACG, CGAACU, CGAAGA, CGAAGC, CGAAGG, CGAAGU, CGAAUA, CGAAUC, CGAAUG, CGAAUU,CGACAA, CGACAC, CGACAU, CGACCA, CGACCU, CGACGA, CGACGC, CGACGG, CGACGU, CGACUA,CGACUG, CGACUU, CGAGAA, CGAGAC, CGAGAG, CGAGAU, CGAGCA, CGAGCC, CGAGCG, CGAGCU,CGAGGC, CGAGGG, CGAGGU, CGAGUA, CGAGUC, CGAGUG, CGAGUU, CGAUAA, CGAUAC, CGAUAG,CGAUAU, CGAUCA, CGAUCC, CGAUCG, CGAUCU, CGAUGA, CGAUGC, CGAUGG, CGAUGU, CGAUUA,CGAUUC, CGAUUG, CGAUUU, CGCAAA, CGCAAC, CGCAAG, CGCAAU, CGCACA, CGCACC, CGCACG,CGCAGA, CGCAGC, CGCAGG, CGCAGU, CGCAUA, CGCAUC, CGCAUG, CGCAUU, CGCCAA, CGCCAC,CGCCAG, CGCCAU, CGCCCA, CGCCCC, CGCCCG, CGCCGA, CGCCGC, CGCCGG, CGCCGU, CGCCUA,CGCCUG, CGCCUU, CGCGAA, CGCGAC, CGCGAG, CGCGAU, CGCGCA, CGCGCC, CGCGCG, CGCGCU,CGCGGA, CGCGGC, CGCGGG, CGCGGU, CGCGUA, CGCGUC, CGCGUG, CGCGUU, CGCUAA, CGCUAC,CGCUAG, CGCUAU, CGCUCA, CGCUCC, CGCUCG, CGCUCU, CGCUGA, CGCUGC, CGCUGG, CGCUGU,CGCUUA, CGCUUC, CGCUUG, CGGAAA, CGGAAC, CGGAAG, CGGACA, CGGACC, CGGACG, CGGACU,CGGAGC, CGGAGG, CGGAGU, CGGAUA, CGGAUU, CGGCAA, CGGCAC, CGGCAG, CGGCCA, CGGCCC,CGGCCG, CGGCGC, CGGCGG, CGGCGU, CGGCUA, CGGCUC, CGGCUG, CGGCUU, CGGGAA, CGGGAC,CGGGAG, CGGGAU, CGGGCA, CGGGCC, CGGGCG, CGGGCU, CGGGGU, CGGGUA, CGGGUC, CGGGUG,CGGUAA, CGGUAC, CGGUAG, CGGUAU, CGGUCA, CGGUCG, CGGUCU, CGGUGA, CGGUGG, CGGUGU,CGGUUA, CGGUUC, CGGUUG, CGGUUU, CGUAAA, CGUAAC, CGUAAG, CGUAAU, CGUACA, CGUACG,CGUACU, CGUAGA, CGUAGC, CGUAGG, CGUAGU, CGUAUA, CGUAUC, CGUAUG, CGUAUU, CGUCAA,CGUCAC, CGUCAG, CGUCAU, CGUCCA, CGUCCC, CGUCCG, CGUCCU, CGUCGA, CGUCGG, CGUCGU,CGUCUA, CGUCUC, CGUCUG, CGUCUU, CGUGAA, CGUGAC, CGUGAG, CGUGAU, CGUGCC, CGUGCG,CGUGCU, CGUGGA, CGUGGG, CGUGGU, CGUGUA, CGUGUG, CGUUAA, CGUUAC, CGUUAG,CGUUAU, CGUUCA, CGUUCC, CGUUCG, CGUUCU, CGUUGA, CGUUGC, CGUUGU, CGUUUA, CGUUUC,CGUUUU, CUAAAA, CUAAAC, CUAAAU, CUAACA, CUAACC, CUAACG, CUAACU, CUAAGA, CUAAGC,CUAAGU, CUAAUA, CUAAUC, CUAAUG, CUACAC, CUACAU, CUACCA, CUACCC, CUACCG, CUACCU,CUACGA, CUACGC, CUACGG, CUACGU, CUACUA, CUACUC, CUACUG, CUAGAA, CUAGAG, CUAGAU,CUAGCA, CUAGCC, CUAGCG, CUAGCU, CUAGGA, CUAGGG, CUAGGU, CUAGUG, CUAGUU, CUAUAA,CUAUAG, CUAUAU, CUAUCA, CUAUCC, CUAUCG, CUAUCU, CUAUGA, CUAUGC, CUAUGG, CUAUGU,CUAUUA, CUAUUG, CUCAAC, CUCAAG, CUCAAU, CUCACC, CUCACG, CUCAGC, CUCAUA, CUCAUC,CUCAUG, CUCAUU, CUCCAC, CUCCCC, CUCCCG, CUCCGA, CUCCGC, CUCCGG, CUCCUA, CUCCUC,CUCCUU, CUCGAA, CUCGAC, CUCGAG, CUCGAU, CUCGCA, CUCGCC, CUCGCG, CUCGGG, CUCGGU,CUCGUA, CUCGUC, CUCGUG, CUCGUU, CUCUAA, CUCUAC, CUCUAU, CUCUCA, CUCUCC, CUCUCU,CUCUGC, CUCUGU, CUCUUA, CUCUUG, CUGAAG, CUGACC, CUGACG, CUGAGC, CUGAUA, CUGAUC,CUGCCG, CUGCCU, CUGCGA, CUGCUA, CUGCUU, CUGGAG, CUGGAU, CUGGCG, CUGGGU, CUGUAC,CUGUCA, CUGUCC, CUGUCG, CUGUGG, CUGUGU, CUGUUA, CUGUUU, CUUAAC, CUUAAG, CUUAAU,CUUACC, CUUACG, CUUAGA, CUUAGC, CUUAGG, CUUAGU, CUUAUA, CUUAUC, CUUAUG, CUUAUU,CUUCAG, CUUCAU, CUUCCA, CUUCCC, CUUCCG, CUUCCU, CUUCGA, CUUCGC, CUUCGG, CUUCGU,CUUCUA, CUUGAC, CUUGAG, CUUGAU, CUUGCA, CUUGCC, CUUGCG, CUUGCU, CUUGGC, CUUGGU,CUUGUU, CUUUAC, CUUUAG, CUUUAU, CUUUCA, CUUUCG, CUUUCU, CUUUGA, CUUUGC, CUUUGU,CUUUUA, CUUUUC, CUUUUG, CUUUUU, GAAAAA, GAAAAG, GAAAAU, GAAACC, GAAACG, GAAAGA,GAAAGC, GAAAGU, GAAAUA, GAAAUC, GAAAUG, GAAAUU, GAACAA, GAACAC, GAACAG, GAACAU,GAACCA, GAACCC, GAACCG, GAACCU, GAACGA, GAACGC, GAACGG, GAACGU, GAACUA, GAACUG,GAACUU, GAAGAC, GAAGAG, GAAGCA, GAAGCG, GAAGCU, GAAGUC, GAAUAA, GAAUAC, GAAUAG,GAAUAU, GAAUCC, GAAUCG, GAAUCU, GAAUGA, GAAUGC, GAAUGU, GAAUUA, GAAUUC, GAAUUU,GACAAA, GACAAG, GACAAU, GACACC, GACAGA, GACAGG, GACAUA, GACAUG, GACAUU, GACCAA,GACCAC, GACCAG, GACCCA, GACCCC, GACCCG, GACCGC, GACCGG, GACCGU, GACCUA, GACCUC,GACCUU, GACGAA, GACGAC, GACGAG, GACGAU, GACGCA, GACGCC, GACGCG, GACGCU, GACGGA,GACGGC, GACGGG, GACGGU, GACGUA, GACGUC, GACGUG, GACGUU, GACUAA, GACUAC, GACUAG,GACUAU, GACUCA, GACUCC, GACUCG, GACUGG, GACUGU, GACUUA, GACUUG, GACUUU, GAGAAU,GAGAGA, GAGAGC, GAGAGG, GAGAUA, GAGAUC, GAGCAA, GAGCAU, GAGCCA, GAGCGA, GAGCGG,GAGCGU, GAGGGU, GAGGUC, GAGGUG, GAGUAA, GAGUAG, GAGUCC, GAGUUC, GAGUUU,GAUAAA, GAUAAC, GAUAAG, GAUAAU, GAUACA, GAUACC, GAUACG, GAUACU, GAUAGA, GAUAGC,GAUAGG, GAUAGU, GAUAUA, GAUCAA, GAUCAC, GAUCAU, GAUCCA, GAUCCC, GAUCCU, GAUCGC,GAUCGG, GAUCGU, GAUCUA, GAUCUG, GAUCUU, GAUGAA, GAUGAC, GAUGAG, GAUGCA, GAUGCC,GAUGCG, GAUGCU, GAUGGC, GAUGGG, GAUGGU, GAUGUG, GAUGUU, GAUUAA, GAUUAC,GAUUAG, GAUUAU, GAUUCA, GAUUCG, GAUUCU, GAUUGA, GAUUGC, GAUUUA, GAUUUC,GAUUUG, GAUUUU, GCAAAC, GCAAAG, GCAAAU, GCAACA, GCAACC, GCAAGC, GCAAGU, GCAAUA,GCAAUC, GCAAUG, GCAAUU, GCACAA, GCACAC, GCACAG, GCACCC, GCACCG, GCACCU, GCACGA,GCACGC, GCACGU, GCACUA, GCACUC, GCACUG, GCACUU, GCAGAU, GCAGCC, GCAGCG, GCAGGC,GCAGUA, GCAGUC, GCAGUG, GCAGUU, GCAUAA, GCAUAG, GCAUAU, GCAUCG, GCAUCU, GCAUGA,GCAUGC, GCAUGG, GCAUGU, GCAUUA, GCAUUC, GCAUUG, GCAUUU, GCCAAA, GCCAAC, GCCAAU,GCCACA, GCCACC, GCCACG, GCCAGA, GCCAGU, GCCAUA, GCCAUC, GCCAUG, GCCAUU, GCCCAA,GCCCAC, GCCCAG, GCCCCG, GCCCGA, GCCCGG, GCCCGU, GCCGAA, GCCGAC, GCCGAG, GCCGAU,GCCGCA, GCCGCU, GCCGGA, GCCGGC, GCCGGG, GCCGGU, GCCGUA, GCCGUC, GCCGUG, GCCGUU,GCCUAA, GCCUAU, GCCUCA, GCCUCC, GCCUCG, GCCUGA, GCCUUA, GCCUUU, GCGAAA, GCGAAC,GCGAAG, GCGAAU, GCGACC, GCGACG, GCGACU, GCGAGA, GCGAGC, GCGAGG, GCGAGU, GCGAUA,GCGAUC, GCGAUG, GCGAUU, GCGCAA, GCGCAC, GCGCAG, GCGCAU, GCGCCA, GCGCCC, GCGCCU,GCGCGA, GCGCGU, GCGCUA, GCGCUC, GCGCUG, GCGCUU, GCGGAA, GCGGAC, GCGGAU, GCGGCA,GCGGCC, GCGGCU, GCGGGA, GCGGUA, GCGGUC, GCGGUU, GCGUAA, GCGUAC, GCGUAG, GCGUAU,GCGUCA, GCGUCC, GCGUCG, GCGUCU, GCGUGA, GCGUGC, GCGUGG, GCGUGU, GCGUUA, GCGUUC,GCGUUG, GCGUUU, GCUAAA, GCUAAC, GCUAAG, GCUAAU, GCUACC, GCUACG, GCUACU, GCUAGA,GCUAGG, GCUAGU, GCUAUA, GCUAUC, GCUAUU, GCUCAA, GCUCAC, GCUCAG, GCUCAU, GCUCCA,GCUCCC, GCUCCG, GCUCGA, GCUCGC, GCUCGU, GCUCUA, GCUCUC, GCUCUU, GCUGAA, GCUGAC,GCUGAU, GCUGCA, GCUGCC, GCUGCG, GCUGCU, GCUGUG, GCUGUU, GCUUAC, GCUUAG, GCUUAU,GCUUCA, GCUUCG, GCUUGA, GCUUGG, GCUUGU, GCUUUA, GCUUUG, GGAAAG, GGAACA, GGAACC,GGAACG, GGAACU, GGAAGU, GGAAUA, GGAAUC, GGAAUU, GGACAA, GGACAC, GGACAG, GGACAU,GGACCG, GGACGA, GGACGC, GGACGU, GGACUA, GGACUC, GGACUU, GGAGAC, GGAGCA, GGAGCG,GGAGGG, GGAGUA, GGAUAA, GGAUAC, GGAUCA, GGAUCC, GGAUCG, GGAUCU, GGAUGC, GGAUUA,GGAUUG, GGCAAU, GGCACA, GGCACU, GGCAGA, GGCAUA, GGCAUC, GGCCAC, GGCCAG, GGCCCC,GGCCGA, GGCCGC, GGCCGU, GGCCUA, GGCCUG, GGCCUU, GGCGAA, GGCGAG, GGCGAU, GGCGCA,GGCGCU, GGCGGU, GGCGUA, GGCGUC, GGCGUG, GGCGUU, GGCUAA, GGCUAC, GGCUAG, GGCUAU,GGCUCC, GGCUCG, GGCUGA, GGCUUA, GGCUUC, GGCUUG, GGGAAU, GGGACA, GGGAGA, GGGAGU,GGGAUA, GGGAUU, GGGCAA, GGGCAC, GGGCAG, GGGCCG, GGGCGG, GGGGCC, GGGGGG,GGGGGU, GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC,GGGUUA, GGGUUG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG,GGUACU, GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC,GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GGUCGG, GGUCGU, GGUCUC,GGUCUU, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC,GGUUAA, GGUUAG, GGUUAU, GGUUCA, GGUUCC, GGUUCG, GGUUGC, GGUUUC, GGUUUU,GUAAAA, GUAAAG, GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU,GUAAUA, GUAAUC, GUAAUG, GUAAUU, GUACAA, GUACAC, GUACAG, GUACAU, GUACCA, GUACCC,GUACCG, GUACCU, GUACGA, GUACGC, GUACGG, GUACGU, GUACUA, GUACUC, GUACUG, GUACUU,GUAGAA, GUAGAC, GUAGCA, GUAGCC, GUAGCG, GUAGCU, GUAGGA, GUAGGC, GUAGGG,GUAGGU, GUAGUA, GUAGUC, GUAUAA, GUAUAC, GUAUAG, GUAUAU, GUAUCA, GUAUCG,GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAUUG, GUAUUU, GUCAAA, GUCAAG,GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG,GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC, GUCCGG, GUCCGU, GUCCUA,GUCCUG, GUCCUU, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU,GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA, GUCGUC, GUCGUU, GUCUAA, GUCUAG, GUCUCA,GUCUCC, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCUUC, GUCUUU, GUGAAA, GUGAAC, GUGAAG,GUGACC, GUGACG, GUGAGA, GUGAGC, GUGAGU, GUGAUC, GUGAUG, GUGAUU, GUGCAC,GUGCAU, GUGCCC, GUGCCG, GUGCGA, GUGCGG, GUGCGU, GUGCUA, GUGCUC, GUGCUG,GUGGAG, GUGGCG, GUGGCU, GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG,GUGUGA, GUGUGC, GUGUGU, GUGUUG, GUGUUU, GUUAAA, GUUAAC, GUUAAG, GUUACA,GUUACC, GUUACG, GUUACU, GUUAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG,GUUAUU, GUUCAA, GUUCAC, GUUCAG, GUUCCA, GUUCCG, GUUCGA, GUUCGC, GUUCGG, GUUCGU,GUUCUA, GUUCUG, GUUGAA, GUUGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA,GUUGGC, GUUGGU, GUUGUC, GUUGUG, GUUGUU, GUUUAA, GUUUAC, GUUUAG, GUUUAU,GUUUCA, GUUUCC, GUUUCU, GUUUGA, GUUUGC, GUUUGG, GUUUGU, GUUUUA, GUUUUC,GUUUUU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU, UAAAGA,UAAAGG, UAAAGU, UAAAUA, UAAAUC, UAAAUG, UAAAUU, UAACAA, UAACAC, UAACAG, UAACCA,UAACCC, UAACCG, UAACCU, UAACGA, UAACGC, UAACGG, UAACGU, UAACUA, UAACUG, UAACUU,UAAGAG, UAAGAU, UAAGCA, UAAGCC, UAAGCG, UAAGCU, UAAGGA, UAAGGC, UAAGGG, UAAGGU,UAAGUA, UAAGUC, UAAGUG, UAAGUU, UAAUAA, UAAUCA, UAAUCC, UAAUCG, UAAUCU, UAAUGA,UAAUGG, UAAUGU, UAAUUA, UAAUUC, UAAUUG, UACAAC, UACAAG, UACAAU, UACACC, UACACG,UACACU, UACAGA, UACAGC, UACAUA, UACAUC, UACAUU, UACCAA, UACCAC, UACCAG, UACCAU,UACCCC, UACCCG, UACCCU, UACCGA, UACCGC, UACCGG, UACCGU, UACCUA, UACCUG, UACGAA,UACGAC, UACGAG, UACGAU, UACGCA, UACGCC, UACGCG, UACGCU, UACGGC, UACGGG, UACGGU,UACGUA, UACGUC, UACGUG, UACGUU, UACUAA, UACUAC, UACUAG, UACUAU, UACUCA, UACUCC,UACUCG, UACUCU, UACUGA, UACUGC, UACUGG, UACUUA, UACUUG, UACUUU, UAGAAA, UAGAAG,UAGAAU, UAGACA, UAGACG, UAGAGA, UAGAGC, UAGAGU, UAGAUA, UAGAUC, UAGAUG, UAGCAU,UAGCCC, UAGCCG, UAGCCU, UAGCGA, UAGCGC, UAGCGU, UAGCUA, UAGCUC, UAGCUG, UAGGAA,UAGGAU, UAGGCG, UAGGCU, UAGGGU, UAGGUC, UAGGUG, UAGGUU, UAGUAA, UAGUAC,UAGUAG, UAGUAU, UAGUCA, UAGUCG, UAGUGU, UAGUUA, UAGUUC, UAGUUG, UAGUUU,UAUAAC, UAUAAG, UAUACU, UAUAGA, UAUAGC, UAUAGG, UAUAGU, UAUAUA, UAUAUC, UAUAUG,UAUAUU, UAUCAA, UAUCAC, UAUCAU, UAUCCA, UAUCCC, UAUCCG, UAUCCU, UAUCGA, UAUCGC,UAUCGG, UAUCGU, UAUCUA, UAUCUC, UAUCUG, UAUCUU, UAUGAA, UAUGAC, UAUGAG,UAUGAU, UAUGCA, UAUGCG, UAUGCU, UAUGGA, UAUGGC, UAUGUC, UAUGUG, UAUGUU,UAUUAG, UAUUCA, UAUUCC, UAUUCG, UAUUCU, UAUUGA, UAUUGG, UAUUUA, UAUUUC,UAUUUG, UAUUUU, UCAAAA, UCAAAC, UCAAAG, UCAACC, UCAACU, UCAAGA, UCAAGC, UCAAUA,UCAAUC, UCAAUG, UCAAUU, UCACCC, UCACCG, UCACCU, UCACGA, UCACGC, UCACGG, UCACGU,UCACUA, UCACUC, UCACUU, UCAGAA, UCAGAC, UCAGAG, UCAGCG, UCAGCU, UCAGGA, UCAGGC,UCAGGU, UCAGUC, UCAGUU, UCAUAA, UCAUCA, UCAUCC, UCAUCG, UCAUGC, UCAUGG, UCAUGU,UCAUUA, UCAUUG, UCCAAA, UCCAAC, UCCAAG, UCCAAU, UCCACA, UCCACC, UCCACG, UCCAGC,UCCAGG, UCCAUA, UCCAUC, UCCAUU, UCCCAA, UCCCAG, UCCCAU, UCCCCC, UCCCCG, UCCCCU,UCCCGA, UCCCGC, UCCCGG, UCCCGU, UCCCUA, UCCCUC, UCCGAA, UCCGAC, UCCGAG, UCCGAU,UCCGCA, UCCGCC, UCCGGA, UCCGGC, UCCGGU, UCCGUA, UCCGUC, UCCGUG, UCCUAA, UCCUCA,UCCUCG, UCCUCU, UCCUGC, UCCUGU, UCCUUA, UCCUUC, UCCUUU, UCGAAA, UCGAAC, UCGAAG,UCGAAU, UCGACA, UCGACC, UCGACG, UCGACU, UCGAGA, UCGAGC, UCGAGG, UCGAUA, UCGAUC,UCGAUG, UCGAUU, UCGCAA, UCGCAC, UCGCAG, UCGCAU, UCGCCA, UCGCCC, UCGCCG, UCGCCU,UCGCGA, UCGCGC, UCGCGU, UCGCUA, UCGCUC, UCGGAA, UCGGAC, UCGGAG, UCGGAU, UCGGCA,UCGGCU, UCGGGG, UCGGGU, UCGGUC, UCGGUG, UCGGUU, UCGUAA, UCGUAC, UCGUAG,UCGUAU, UCGUCA, UCGUCC, UCGUCG, UCGUCU, UCGUGA, UCGUGU, UCGUUA, UCGUUC, UCGUUG,UCGUUU, UCUAAC, UCUAAG, UCUAAU, UCUACA, UCUACC, UCUACG, UCUACU, UCUAGC, UCUAGG,UCUAGU, UCUAUA, UCUAUC, UCUAUG, UCUAUU, UCUCAG, UCUCAU, UCUCCG, UCUCGC, UCUCGG,UCUCGU, UCUCUC, UCUGAA, UCUGAU, UCUGCA, UCUGCG, UCUGCU, UCUGGC, UCUGGU, UCUGUC,UCUGUG, UCUGUU, UCUUAA, UCUUAC, UCUUAG, UCUUAU, UCUUCA, UCUUCC, UCUUCG, UCUUCU,UCUUGC, UCUUGG, UCUUGU, UCUUUA, UCUUUC, UCUUUG, UCUUUU, UGAAAA, UGAAAC,UGAACA, UGAACC, UGAAGG, UGAAUC, UGAAUG, UGACAA, UGACAC, UGACAG, UGACCA, UGACCC,UGACCG, UGACGA, UGACGC, UGACGG, UGACGU, UGACUA, UGACUC, UGACUU, UGAGAG, UGAGAU,UGAGCA, UGAGCC, UGAGCU, UGAGGC, UGAGGU, UGAGUA, UGAGUU, UGAUAC, UGAUAG,UGAUAU, UGAUCA, UGAUCG, UGAUCU, UGAUGA, UGAUGC, UGAUGG, UGAUGU, UGAUUA,UGAUUC, UGAUUG, UGAUUU, UGCAAC, UGCAAG, UGCACA, UGCACG, UGCAGG, UGCAGU, UGCAUC,UGCCCA, UGCCCC, UGCCCG, UGCCGA, UGCCGC, UGCCGG, UGCCGU, UGCCUA, UGCCUC, UGCCUG,UGCCUU, UGCGAA, UGCGAC, UGCGAU, UGCGCC, UGCGCG, UGCGCU, UGCGGC, UGCGGG, UGCGGU,UGCGUA, UGCGUC, UGCGUG, UGCGUU, UGCUAC, UGCUAU, UGCUCC, UGCUCG, UGCUGC, UGCUGG,UGCUGU, UGCUUA, UGCUUU, UGGAAC, UGGAAG, UGGAGC, UGGAUC, UGGAUU, UGGCAA,UGGCAC, UGGCAG, UGGCCG, UGGCCU, UGGCGA, UGGCGC, UGGCGU, UGGCUA, UGGCUC, UGGCUU,UGGGAA, UGGGCA, UGGGCC, UGGGGC, UGGGUC, UGGUAA, UGGUAG, UGGUAU, UGGUCC,UGGUCG, UGGUCU, UGGUGA, UGGUGC, UGGUGG, UGGUGU, UGGUUA, UGGUUG, UGUAAA,UGUAAC, UGUAAG, UGUACC, UGUACG, UGUACU, UGUAGA, UGUAGC, UGUAGU, UGUAUC,UGUAUU, UGUCAA, UGUCAC, UGUCAG, UGUCAU, UGUCCA, UGUCCC, UGUCCG, UGUCGA, UGUCGC,UGUCGG, UGUCGU, UGUCUA, UGUCUC, UGUGAC, UGUGAG, UGUGAU, UGUGCA, UGUGGU,UGUGUA, UGUGUU, UGUUAC, UGUUAG, UGUUAU, UGUUCA, UGUUCC, UGUUCG, UGUUGG,UGUUGU, UGUUUA, UGUUUC, UGUUUG, UGUUUU, UUAAAA, UUAAAC, UUAAAG, UUAAAU,UUAACC, UUAACG, UUAACU, UUAAGU, UUAAUA, UUAAUC, UUAAUG, UUAAUU, UUACAA, UUACAC,UUACAG, UUACAU, UUACCA, UUACCC, UUACCG, UUACCU, UUACGA, UUACGC, UUACGG, UUACGU,UUACUA, UUACUC, UUACUG, UUACUU, UUAGAA, UUAGAC, UUAGCC, UUAGCG, UUAGCU, UUAGGC,UUAGGU, UUAGUA, UUAGUC, UUAGUU, UUAUAA, UUAUAC, UUAUAG, UUAUAU, UUAUCC,UUAUCG, UUAUCU, UUAUGA, UUAUGG, UUAUGU, UUAUUA, UUAUUC, UUAUUG, UUAUUU,UUCAAC, UUCAAU, UUCACA, UUCACC, UUCACG, UUCACU, UUCAGC, UUCAGG, UUCAGU, UUCAUA,UUCAUC, UUCAUG, UUCAUU, UUCCAA, UUCCCA, UUCCCG, UUCCGA, UUCCGU, UUCCUU, UUCGAA,UUCGAC, UUCGAG, UUCGAU, UUCGCA, UUCGCC, UUCGCG, UUCGCU, UUCGGA, UUCGGC, UUCGGG,UUCGGU, UUCGUA, UUCGUC, UUCGUG, UUCGUU, UUCUAC, UUCUAG, UUCUCA, UUCUCG,UUCUGG, UUCUUA, UUCUUU, UUGAAA, UUGAAC, UUGAAG, UUGAAU, UUGACC, UUGACG,UUGACU, UUGAGA, UUGAGC, UUGAGU, UUGAUA, UUGAUC, UUGAUG, UUGAUU, UUGCAA,UUGCAC, UUGCAG, UUGCAU, UUGCCC, UUGCCG, UUGCGA, UUGCGC, UUGCGG, UUGCGU, UUGCUA,UUGCUC, UUGCUG, UUGCUU, UUGGAA, UUGGAG, UUGGCC, UUGGCG, UUGGCU, UUGGGC,UUGGGU, UUGGUA, UUGGUG, UUGUAA, UUGUAC, UUGUCA, UUGUCG, UUGUCU, UUGUGC,UUGUGG, UUGUUA, UUGUUG, UUGUUU, UUUAAA, UUUAAC, UUUAAG, UUUAAU, UUUACA,UUUACC, UUUACG, UUUACU, UUUAGA, UUUAGC, UUUAGG, UUUAGU, UUUAUA, UUUAUC,UUUAUG, UUUAUU, UUUCAU, UUUCCA, UUUCCG, UUUCCU, UUUCGA, UUUCGC, UUUCGG,UUUCGU, UUUCUA, UUUCUC, UUUCUG, UUUCUU, UUUGAA, UUUGAC, UUUGAG, UUUGAU,UUUGCC, UUUGCU, UUUGGA, UUUGGC, UUUGGG, UUUGGU, UUUGUA, UUUGUC, UUUGUU,UUUUAA, UUUUAG, UUUUAU, UUUUCC, UUUUCG, UUUUCU, UUUUGA, UUUUGC, UUUUGG,UUUUGU, UUUUUA, UUUUUC, UUUUUU

TABLE 2 Oligonucleotide sequences made for testing Oligo Gene Expt CellAssay Name RQ RQ SE Name Type Line/Tissue [Oligo] Type Coordinates_gSMN1- 0.812671952 0.135251351 SMN1 in vitro RPTEC 100 qRTPCRSMN1:21157U20 01 SMN1- 0.857032101 0.027318737 SMN1 in vitro RPTEC 50qRTPCR SMN1:21157U20 01 SMN1- 0.167998915 0.167998672 SMN1 in vitroHep3B 50 qRTPCR SMN1:21157U20 01 SMN1- 1.048125302 0.039302784 SMN1 invitro Hep3B 100 qRTPCR SMN1:21157U20 01 SMN1- 1.381704207 0.053290565SMN1 in vitro Hep3B 10 qRTPCR SMN1:21157U20 01 SMN1- 0.9798692470.020515227 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21157U20 01 SMN1-0.760000318 0.042993212 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21158U20 02SMN1- 0.987138447 0.068187998 SMN1 in vitro RPTEC 50 qRTPCRSMN1:21158U20 02 SMN1- 2.252494526 1.803190669 SMN1 in vitro Hep3B 50qRTPCR SMN1:21158U20 02 SMN1- 1.114387973 0.026733251 SMN1 in vitroHep3B 100 qRTPCR SMN1:21158U20 02 SMN1- 1.34641929 0.027641281 SMN1 invitro Hep3B 10 qRTPCR SMN1:21158U20 02 SMN1- 1.153697083 0.024999991SMN1 in vitro Hep3B 30 qRTPCR SMN1:21158U20 02 SMN1- 1.907229750.525939296 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21159U20 03 SMN1-1.132758264 0.094640177 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21159U20 03SMN1- 0.29619174 0.173282309 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21159U2003 SMN1- 1.48817935 0.172719507 SMN1 in vitro Hep3B 100 qRTPCRSMN1:21159U20 03 SMN1- 1.29932826 0.059825228 SMN1 in vitro Hep3B 10qRTPCR SMN1:21159U20 03 SMN1- 1.511567814 0.054178175 SMN1 in vitroHep3B 30 qRTPCR SMN1:21159U20 03 SMN1- 1.048306517 0.243934543 SMN1 invitro RPTEC 100 qRTPCR SMN1:21160U20 04 SMN1- 1.322407267 0.100022392SMN1 in vitro RPTEC 50 qRTPCR SMN1:21160U20 04 SMN1- 0.1331700130.032824391 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21160U20 04 SMN1-1.289550163 0.330195987 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21160U20 04SMN1- 1.280225492 0.062577972 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21160U20 04 SMN1- 1.488482795 0.044641287 SMN1 in vitro Hep3B 30qRTPCR SMN1:21160U20 04 SMN1- 0.876747527 0.087392504 SMN1 in vitroRPTEC 100 qRTPCR SMN1:21161U20 05 SMN1- 1.167120345 0.069814091 SMN1 invitro RPTEC 50 qRTPCR SMN1:21161U20 05 SMN1- 0.088317863 0.039887014SMN1 in vitro Hep3B 50 qRTPCR SMN1:21161U20 05 SMN1- 1.3100532560.234231348 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21161U20 05 SMN1-1.038699643 0.056421362 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21161U20 05SMN1- 0.859144751 0.039970015 SMN1 in vitro Hep3B 30 qRTPCRSMN1:21161U20 05 SMN1- 0.704659891 0.087244119 SMN1 in vitro RPTEC 100qRTPCR SMN1:21162U20 06 SMN1- 1.11194006 0.088571377 SMN1 in vitro RPTEC50 qRTPCR SMN1:21162U20 06 SMN1- 0.57685962 0.246186541 SMN1 in vitroHep3B 50 qRTPCR SMN1:21162U20 06 SMN1- 1.419418884 0.432447122 SMN1 invitro Hep3B 100 qRTPCR SMN1:21162U20 06 SMN1- 1.146251704 0.051891541SMN1 in vitro Hep3B 10 qRTPCR SMN1:21162U20 06 SMN1- 1.0306823170.013070835 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21162U20 06 SMN1-0.682085732 0.084885351 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21163U20 07SMN1- 0.975853552 0.034178542 SMN1 in vitro RPTEC 50 qRTPCRSMN1:21163U20 07 SMN1- 1.013252314 0.118540759 SMN1 in vitro Hep3B 50qRTPCR SMN1:21163U20 07 SMN1- 1.039381902 0.059815387 SMN1 in vitroHep3B 100 qRTPCR SMN1:21163U20 07 SMN1- 1.156949605 0.107385405 SMN1 invitro Hep3B 10 qRTPCR SMN1:21163U20 07 SMN1- 1.239503954 0.134603844SMN1 in vitro Hep3B 30 qRTPCR SMN1:21163U20 07 SMN1- 0.9487148880.142708231 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21164U20 08 SMN1-1.312080445 0.058464993 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21164U20 08SMN1- 0.216530007 0.177400555 SMN1 in vitro Hep3B 50 qRTPCRSMN1:21164U20 08 SMN1- 2.082151781 0.815184252 SMN1 in vitro Hep3B 100qRTPCR SMN1:21164U20 08 SMN1- 1.010090604 0.200588791 SMN1 in vitroHep3B 10 qRTPCR SMN1:21164U20 08 SMN1- 1.223947667 0.295307243 SMN1 invitro Hep3B 30 qRTPCR SMN1:21164U20 08 SMN1- 0.77519063 0.098695118 SMN1in vitro RPTEC 100 qRTPCR SMN1:21165U20 09 SMN1- 1.685731616 0.014884028SMN1 in vitro RPTEC 50 qRTPCR SMN1:21165U20 09 SMN1- 0.6214067810.227211261 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21165U20 09 SMN1-0.85593922 0.256108337 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21165U20 09SMN1- 0.940186097 0.197008464 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21165U20 09 SMN1- 0.864481145 0.162739271 SMN1 in vitro Hep3B 30qRTPCR SMN1:21165U20 09 SMN1- 0.945730986 0.08072952 SMN1 in vitro RPTEC100 qRTPCR SMN1:21166U20 10 SMN1- 1.574526902 0.123062684 SMN1 in vitroRPTEC 50 qRTPCR SMN1:21166U20 10 SMN1- 0.482822242 0.131557474 SMN1 invitro Hep3B 50 qRTPCR SMN1:21166U20 10 SMN1- 1.280629128 0.17088425 SMN1in vitro Hep3B 100 qRTPCR SMN1:21166U20 10 SMN1- 1.127254654 0.152486374SMN1 in vitro Hep3B 10 qRTPCR SMN1:21166U20 10 SMN1- 1.0695714580.122106758 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21166U20 10 SMN1-0.774436979 0.038076182 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21167U20 11SMN1- 1.562714254 0.158043098 SMN1 in vitro RPTEC 50 qRTPCRSMN1:21167U20 11 SMN1- 0.463655938 0.295513886 SMN1 in vitro Hep3B 50qRTPCR SMN1:21167U20 11 SMN1- 0.957611652 0.334137541 SMN1 in vitroHep3B 100 qRTPCR SMN1:21167U20 11 SMN1- 1.225973818 0.223472758 SMN1 invitro Hep3B 10 qRTPCR SMN1:21167U20 11 SMN1- 1.089302259 0.126414268SMN1 in vitro Hep3B 30 qRTPCR SMN1:21167U20 11 SMN1- 0.9814294760.07937384 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21168U20 12 SMN1-1.585088128 0.05291912 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21168U20 12SMN1- 0.208586047 0.187017655 SMN1 in vitro Hep3B 50 qRTPCRSMN1:21168U20 12 SMN1- 3.266965896 2.002074369 SMN1 in vitro Hep3B 100qRTPCR SMN1:21168U20 12 SMN1- 1.03381379 0.204376291 SMN1 in vitro Hep3B10 qRTPCR SMN1:21168U20 12 SMN1- 1.137471671 0.246791954 SMN1 in vitroHep3B 30 qRTPCR SMN1:21168U20 12 SMN1- 0.749636437 0.103277003 SMN1 invitro RPTEC 100 qRTPCR SMN1:21169U20 13 SMN1- 1.175989263 0.122355585SMN1 in vitro RPTEC 50 qRTPCR SMN1:21169U20 13 SMN1- 0.1614991590.079356287 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21169U20 13 SMN1-1.287763591 0.090306717 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21169U20 13SMN1- 1.336851675 0.1778149 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21169U2013 SMN1- 1.037772291 0.039404507 SMN1 in vitro Hep3B 30 qRTPCRSMN1:21169020 13 SMN1- 0.771635177 0.086041959 SMN1 in vitro RPTEC 100qRTPCR SMN1:21170U20 14 SMN1- 1.467048548 0.073113884 SMN1 in vitroRPTEC 50 qRTPCR SMN1:21170U20 14 SMN1- 1.978254154 1.352951156 SMN1 invitro Hep3B 50 qRTPCR SMN1:21170U20 14 SMN1- 1.311990937 0.073121634SMN1 in vitro Hep3B 100 qRTPCR SMN1:21170U20 14 SMN1- 1.128927770.147162701 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21170U20 14 SMN1-0.855795121 0.017797181 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21170U20 14SMN1- 0.891491964 0.039822032 SMN1 in vitro RPTEC 100 qRTPCRSMN1:21171U20 15 SMN1- 1.573440342 0.117453017 SMN1 in vitro RPTEC 50qRTPCR SMN1:21171U20 15 SMN1- 0.366043104 0.117162019 SMN1 in vitroHep3B 50 qRTPCR SMN1:21171U20 15 SMN1- 1.738217394 0.520148155 SMN1 invitro Hep3B 100 qRTPCR SMN1:21171U20 15 SMN1- 1.383201337 0.101830776SMN1 in vitro Hep3B 10 qRTPCR SMN1:21171U20 15 SMN1- 1.6194950520.038364989 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21171U20 15 SMN1-0.73721881 0.038067583 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21172U20 16SMN1- 1.441616196 0.059823944 SMN1 in vitro RPTEC 50 qRTPCRSMN1:21172U20 16 SMN1- 0.510056605 0.286522659 SMN1 in vitro Hep3B 50qRTPCR SMN1:21172U20 16 SMN1- 1.381914214 0.247880229 SMN1 in vitroHep3B 100 qRTPCR SMN1:21172U20 16 SMN1- 1.310073573 0.026347093 SMN1 invitro Hep3B 10 qRTPCR SMN1:21172U20 16 SMN1- 1.418132646 0.082371708SMN1 in vitro Hep3B 30 qRTPCR SMN1:21172U20 16 SMN1- 1.2190656510.281987674 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21173U20 17 SMN1-1.274819195 0.179527293 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21173U20 17SMN1- 0.416739222 0.066066242 SMN1 in vitro Hep3B 50 qRTPCRSMN1:21173U20 17 SMN1- 3.331843017 0.970174873 SMN1 in vitro Hep3B 100qRTPCR SMN1:21173U20 17 SMN1- 1.260856522 0.038565799 SMN1 in vitroHep3B 10 qRTPCR SMN1:21173U20 17 SMN1- 1.609045311 0.10487434 SMN1 invitro Hep3B 30 qRTPCR SMN1:21173U20 17 SMN1- 0.868441941 0.088184698SMN1 in vitro RPTEC 100 qRTPCR SMN1:21174U20 18 SMN1- 1.2216635740.064445539 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21174U20 18 SMN1-10.28455167 3.929310832 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21174U20 18SMN1- 1.800920764 0.42559045 SMN1 in vitro Hep3B 100 qRTPCRSMN1:21174U20 18 SMN1- 1.261752602 0.069817143 SMN1 in vitro Hep3B 10qRTPCR SMN1:21174U20 18 SMN1- 1.592700796 0.199280916 SMN1 in vitroHep3B 30 qRTPCR SMN1:21174U20 18 SMN1- 0.705512452 0.06496675 SMN1 invitro RPTEC 100 qRTPCR SMN1:21175U20 19 SMN1- 1.43433309 0.075936965SMN1 in vitro RPTEC 50 qRTPCR SMN1:21175U20 19 SMN1- 0.5389321560.309273594 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21175U20 19 SMN1-1.17374637 0.179415746 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21175U20 19SMN1- 1.186141471 0.036729063 SMN1 in vitro Hep38 10 qRTPCRSMN1:21175U20 19 SMN1- 1.834775368 0.155761723 SMN1 in vitro Hep3B 30qRTPCR SMN1:21175U20 19 SMN1- 0.826303453 0.062998254 SMN1 in vitroRPTEC 100 qRTPCR SMN1:21176U20 20 SMN1- 1.505786689 0.170697984 SMN1 invitro RPTEC 50 qRTPCR SMN1:21176U20 20 SMN1- 0.06244992 0.049069571 SMN1in vitro Hep3B 50 qRTPCR SMN1:21176U20 20 SMN1- 1.541480855 0.461158669SMN1 in vitro Hep3B 100 qRTPCR SMN1:21176U20 20 SMN1- 1.0899856920.043750568 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21176U20 20 SMN1-1.41531375 0.146502726 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21176U20 20SMN1- 0.865566453 0.209455026 SMN1 in vitro RPTEC 100 qRTPCRSMN1:21177U20 21 SMN1- 1.466688787 0.116267764 SMN1 in vitro RPTEC 50qRTPCR SMN1:21177U20 21 SMN1- 0.388233514 0.139680869 SMN1 in vitroHep3B 50 qRTPCR SMN1:21177U20 21 SMN1- 1.366269447 0.239420557 SMN1 invitro Hep3B 100 qRTPCR SMN1:21177U20 21 SMN1- 1.354554841 0.013175463SMN1 in vitro Hep3B 10 qRTPCR SMN1:21177U20 21 SMN1- 2.0269683820.27827902 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21177U20 21 SMN1-0.639934851 0.011679891 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21178U20 22SMN1- 1.242593923 0.02840519 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21178U2022 SMN1- 0.229857922 0.128101282 SMN1 in vitro Hep3B 50 qRTPCRSMN1:21178U20 22 SMN1- 1.499722255 0.568788539 SMN1 in vitro Hep3B 100qRTPCR SMN1:21178U20 22 SMN1- 1.234783764 0.017119432 SMN1 in vitroHep3B 10 qRTPCR SMN1:21178U20 22 SMN1- 1.509695591 0.175764156 SMN1 invitro Hep3B 30 qRTPCR SMN1:21178U20 22 SMN1- 0.748031845 0.083732479SMN1 in vitro RPTEC 100 qRTPCR SMN1:21179U20 23 SMN1- 1.339109730.070877143 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21179U20 23 SMN1-0.384143384 0.14723735 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21179U20 23SMN1- 2.620195611 0.342101826 SMN1 in vitro Hep3B 100 qRTPCRSMN1:21179U20 23 SMN1- 1.473663866 0.053762605 SMN1 in vitro Hep3B 10qRTPCR SMN1:21179U20 23 SMN1- 1.920800418 0.127336842 SMN1 in vitroHep3B 30 qRTPCR SMN1:21179U20 23 SMN1- 0.907436601 0.24201681 SMN1 invitro RPTEC 100 qRTPCR SMN1:21180U20 24 SMN1- 1.28379369 0.158661709SMN1 in vitro RPTEC 50 qRTPCR SMN1:21180U20 24 SMN1- 0.9631002080.117802422 SMN1 in vitro Hep3B 50 qRTPCR SMN1:21180U20 24 SMN1-0.994753299 0.268415648 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21180U20 24SMN1- 0.965440348 0.032646295 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21180U20 24 SMN1- 1.140566171 0.10163121 SMN1 in vitro Hep3B 30qRTPCR SMN1:21180U20 24 SMN1- 0.908854808 0.076026035 SMN1 in vitroRPTEC 100 qRTPCR SMN1:21181U20 25 SMN1- 1.226185041 0.044422705 SMN1 invitro RPTEC 50 qRTPCR SMN1:21181U20 25 SMN1- 1.055082301 0.326768036SMN1 in vitro Hep3B 50 qRTPCR SMN1:21181U20 25 SMN1- 0.9691850380.226484866 SMN1 in vitro Hep3B 100 qRTPCR SMN1:21181U20 25 SMN1-1.00974636 0.122120737 SMN1 in vitro Hep38 10 qRTPCR SMN1:21181U20 25SMN1- 1.081303639 0.101827303 SMN1 in vitro Hep3B 30 qRTPCRSMN1:21181U20 25 SMN1- 0.876444072 0.070457909 SMN1 in vitro RPTEC 100qRTPCR SMN1:21182U20 26 SMN1- 1.632434888 0.061512357 SMN1 in vitroRPTEC 50 qRTPCR SMN1:21182U20 26 SMN1- 0.071593319 0.071592884 SMN1 invitro Hep3B 50 qRTPCR SMN1:21182U20 26 SMN1- 1.999202516 0.420387669SMN1 in vitro Hep38 100 qRTPCR SMN1:21182U20 26 SMN1- 0.9741075840.066863661 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21182U20 26 SMN1-1.030227891 0.096105098 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21182U20 26SMN1- 0.834365703 0.108102871 SMN1 in vitro RPTEC 100 qRTPCRSMN1:21157U15 27 SMN1- 1.589954219 0.093101653 SMN1 in vitro RPTEC 50qRTPCR SMN1:21157U15 27 SMN1- 0.747186714 0.007807701 SMN1 in vitroHep3B 30 qRTPCR SMN1:21157U15 27 SMN1- 1.049068744 0.092645193 SMN1 invitro Hep3B 10 qRTPCR SMN1:21157U15 27 SMN1- 1.058343694 0.208931576SMN1 in vitro RPTEC 100 qRTPCR SMN1:21158U15 28 SMN1- 1.4023484140.101950771 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21158U15 28 SMN1-1.150224316 0.080077707 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21158U15 28SMN1- 1.219828396 0.031782762 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21158U15 28 SMN1- 0.712268587 0.077572838 SMN1 in vitro RPTEC 100qRTPCR SMN1:21159U15 29 SMN1- 1.145305552 0.044575389 SMN1 in vitroRPTEC 50 qRTPCR SMN1:21159U15 29 SMN1- 0.937393865 0.015700783 SMN1 invitro Hep3B 30 qRTPCR SMN1:21159U15 29 SMN1- 1.208521962 0.084021899SMN1 in vitro Hep3B 10 qRTPCR SMN1:21159U15 29 SMN1- 0.8695041090.147682779 SMN1 in vitro RPTEC 100 qRTPCR SMN1:21160U15 30 SMN1-1.166995709 0.128900531 SMN1 in vitro RPTEC 50 qRTPCR SMN1:21160U15 30SMN1- 1.069533423 0.042258392 SMN1 in vitro Hep3B 30 qRTPCRSMN1:21160U15 30 SMN1- 1.004618999 0.068245537 SMN1 in vitro Hep3B 10qRTPCR SMN1:21160U15 30 SMN1- 1.223685297 0.155258366 SMN1 in vitroHep3B 30 qRTPCR SMN1:21161U15 31 SMN1- 0.936569575 0.083367899 SMN1 invitro Hep3B 10 qRTPCR SMN1:21161U15 31 SMN1- 1.032978469 0.02312057 SMN1in vitro Hep3B 30 qRTPCR SMN1:21162U15 32 SMN1- 1.053045821 0.030158389SMN1 in vitro Hep3B 10 qRTPCR SMN1:21162U15 32 SMN1- 1.0463614070.038971809 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21163U15 33 SMN1-1.233302232 0.063255341 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21163U15 33SMN1- 1.079876751 0.09859402 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21164U1534 SMN1- 1.271026183 0.067019476 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21164U15 34 SMN1- 0.861464008 0.024095912 SMN1 in vitro Hep3B 30qRTPCR SMN1:21165U15 35 SMN1- 0.836966392 0.054159619 SMN1 in vitroHep3B 10 qRTPCR SMN1:21165U15 35 SMN1- 1.26636324 0.046963681 SMN1 invitro Hep3B 30 qRTPCR SMN1:21166U15 36 SMN1- 1.326257117 0.039674649SMN1 in vitro Hep3B 10 qRTPCR SMN1:21166U15 36 SMN1- 1.2326900860.043476252 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21167U15 37 SMN11.144632987 0.058433353 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21167U15 37SMN1- 0.843241863 0.033808043 SMN1 in vitro Hep3B 30 qRTPCRSMN1:21168U15 38 SMN1- 0.93818033 0.011376217 SMN1 in vitro Hep3B 10qRTPCR SMN1:21168U15 38 SMN1- 0.663746249 0.045527014 SMN1 in vitroHep3B 30 qRTPCR SMN1:21169U15 39 SMN1- 0.891764551 0.019395327 SMN1 invitro Hep3B 10 qRTPCR SMN1:21169U15 39 SMN1- 0.888138653 0.081401804SMN1 in vitro Hep3B 30 qRTPCR SMN1:21170U15 40 SMN1- 0.8716028990.065372936 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21170U15 40 SMN1-0.882466148 0.031016749 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21171U15 41SMN1- 1.093694765 0.025996502 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21171U15 41 SMN1- 0.956860836 0.043558382 SMN1 in vitro Hep3B 30qRTPCR SMN1:21172U15 42 SMN1- 1.151755999 0.067662107 SMN1 in vitroHep3B 10 qRTPCR SMN1:21172U15 42 SMN1- 1.341919782 0.08080776 SMN1 invitro Hep3B 30 qRTPCR SMN1:21173U15 43 SMN1- 1.692919815 0.084669198SMN1 in vitro Hep3B 10 qRTPCR SMN1:21173U15 43 SMN1- 0 0 SMN1 NA NA 0 NASMN1:21174U15 44 SMN1- 1.807236897 0.11410948 SMN1 in vitro Hep3B 30qRTPCR SMN1:21175U15 45 SMN1- 1.377773703 0.108540058 SMN1 in vitroHep3B 10 qRTPCR SMN1:21175U15 45 SMN1- 1.545649538 0.064006814 SMN1 invitro Hep3B 30 qRTPCR SMN1:21176U15 46 SMN1- 1.354291504 0.038498944SMN1 in vitro Hep3B 10 qRTPCR SMN1:21176U15 46 SMN1- 2.7115983610.260043446 SMN1 in vitro Hep38 30 qRTPCR SMN1:21177U15 47 SMN1-1.986674786 0.119436675 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21177U15 47SMN1- 1.482342195 0.063036343 SMN1 in vitro Hep3B 30 qRTPCRSMN1:21178U15 48 SMN1- 2.597350628 0.145439801 SMN1 in vitro Hep3B 10qRTPCR SMN1:21178U15 48 SMN1- 1.534493905 0.110688365 SMN1 in vitroHep3B 30 qRTPCR SMN1:21179U15 49 SMN1- 2.223340784 0.148702992 SMN1 invitro Hep3B 10 qRTPCR SMN1:21179U15 49 SMN1- 0.897421396 0.034254931SMN1 in vitro Hep3B 30 qRTPCR SMN1:21180U15 50 SMN1- 1.1323627810.078523003 SMN1 in vitro Hep3B 10 qRTPCR SMN1:21180U15 50 SMN1-1.157921368 0.044256319 SMN1 in vitro Hep3B 30 qRTPCR SMN1:21181U15 51SMN1- 1.177604665 0.038060353 SMN1 in vitro Hep3B 10 qRTPCRSMN1:21181U15 51 SMN1- 0.973548353 0.051461583 SMN1 in vitro Hep3B 30qRTPCR SMN1:21182U15 52 SMN1- 1.068355642 0.060851146 SMN1 in vitroHep3B 10 qRTPCR SMN1:21182U15 52

TABLE 3 Oligonucleotide Modifications Symbol Feature Description bio 5′biotin dAs DNA w/3′ thiophosphate dCs DNA w/3′ thiophosphate dGs DNAw/3′ thiophosphate dTs DNA w/3′ thiophosphate dG DNA enaAs ENA w/3′thiophosphate enaCs ENA w/3′ thiophosphate enaGs ENA w/3′ thiophosphateenaTs ENA w/3′ thiophosphate fluAs 2′-fluoro w/3′ thiophosphate fluCs2′-fluoro w/3′ thiophosphate fluGs 2′-fluoro w/3′ thiophosphate fluUs2′-fluoro w/3′ thiophosphate InaAs LNA w/3′ thiophosphate InaCs LNA w/3′thiophosphate InaGs LNA w/3′ thiophosphate InaTs LNA w/3′ thiophosphateomeAs 2′-OMe w/3′ thiophosphate omeCs 2′-OMe w/3′ thiophosphate omeGs2′-OMe w/3′ thiophosphate omeTs 2′-OMe w/3′ thiophosphate InaAs-Sup LNAw/3′ thiophosphate at 3′ terminus InaCs-Sup LNA w/3′ thiophosphate at 3′terminus InaGs-Sup LNA w/3′ thiophosphate at 3′ terminus InaTs-Sup LNAw/3′ thiophosphate at 3′ terminus InaA-Sup LNA w/3′ OH at 3′ terminusInaC-Sup LNA w/3′ OH at 3′ terminus InaG-Sup LNA w/3′ OH at 3′ terminusInaT-Sup LNA w/3′ OH at 3′ terminus omeA-Sup 2′-OMe w/3′ OH at 3′terminus omeC-Sup 2′-OMe w/3′ OH at 3′ terminus omeG-Sup 2′-OMe w/3′ OHat 3′ terminus omeU-Sup 2′-OMe w/3′ OH at 3′ terminus dAs-Sup DNA w/3′thiophosphate at 3′ terminus dCs-Sup DNA w/3′ thiophosphate at 3′terminus dGs-Sup DNA w/3′ thiophosphate at 3′ terminus dTs-Sup DNA w/3′thiophosphate at 3′ terminus dA-Sup DNA w/3′ OH at 3′ terminus dC-SupDNA w/3′ OH at 3′ terminus dG-Sup DNA w/3′ OH at 3′ terminus dT-Sup DNAw/3′ OH at 3′ terminus

BRIEF DESCRIPTION OF SEQUENCE LISTING

SeqID Chrom Gene Chrom Start Chrom End Strand Name 1 chr5 SMN1 7020876870260838 + human SMN1 2 chr5 SMN2 69333350 69385422 + human SMN2 3 chr9SMNP 20319406 20344375 + human SMNP 4 chr5 SMN1 70208768 70260838 −human SMN1_revComp 5 chr5 SMN2 69333350 69385422 − human SMN2_revComp 6chr9 SMNP 20319406 20344375 − human SMNP_revComp 7 chr13 Smn1 100881160100919653 + mouse Smn1 8 chr13 Smn1 100881160 100919653 − mouseSmn1_revComp 9 chr5 SMN1 70240095 70240127 − S48-193240 9 chr5 SMN269364672 69364704 + S48-193240 10 chr5 SMN1 70214393 70214822 +S48-441814 10 chr5 SMN2 69338976 69339405 + S48-441814 11 chr5 SMN170214064 70214108 + S48-441815 11 chr5 SMN2 69338647 69338691 +S48-441815 12 chr5 SMN1 70214276 70214317 + S48-473289 12 chr5 SMN269338859 69338900 + S48-473289 13 chr5 SMN1 70214445 70214472 +S48-473290 13 chr5 SMN2 69339028 69339055 + S48-473290 14 chr5 SMN170238095 70242127 + S48-193240 + 2K 15 chr5 SMN2 69362672 69366704 +S48-193240 + 2K 16 chr5 SMN1 70212393 70216822 + S48-441814 + 2K 17 chr5SMN2 69336976 69341405 + S48-441814 + 2K 18 chr5 SMN1 7021206470216108 + S48-441815 + 2K 19 chr5 SMN2 69336647 69340691 + S48-441815 +2K 20 chr5 SMN1 70212276 70216317 + S48-473289 + 2K 21 chr5 SMN269336859 69340900 + S48-473289 + 2K 22 chr5 SMN1 70212445 70216472 +S48-473290 + 2K 23 chr5 SMN2 69337028 69341055 + S48-473290 + 2K 24 chr5SMN1 70240510 70240551 − S48-193241 24 chr5 SMN2 69365087 69365128 −S48-193241 25 chr5 SMN1 70241924 70241968 − S48-193242 25 chr5 SMN269366499 69366543 − S48-193242 26 chr5 SMN1 70238510 70242551 −S48-193241 + 2K 27 chr5 SMN2 69363087 69367128 − S48-193241 + 2K 28 chr5SMN1 70239924 70243968 − S48-193242 + 2K 29 chr5 SMN2 69364499 69368543− S48-193242 + 2K 13100 chr5 SMN1 70247831 70247845 + Splice controlsequence 13100 chr5 SMN2 69372411 69372425 + Splice control sequence13101 chr5 SMN2 69372402 69372845 + Intron 7Single Strand Oligonucleotides (Antisense Strand of Target Gene)SeqID range: 30 to 8329, 13088-13094SeqIDs w/o G Runs:30-142, 156-560, 575-780, 794-912, 926-1013, 1027-1078, 1092-1286,1300-1335, 1349-1385, 1399-1453, 1460-1527, 1548-1555, 1571-1653,1675-1691, 1706-1802, 1816-1883, 1897-2009, 2023-2141, 2165-2289,2303-2320, 2334-2447, 2461-2494, 2508-2526, 2540-2545, 2571-2635,2651-2670, 2689-2763, 2772-2814, 2828-2854, 2868-3030, 3044-3256,3270-3360, 3374-3400, 3414-3722, 3737, 3759-3783, 3797-3970, 3986-4059,4073-4153, 4175-4240, 4255-4415, 4438-4441, 4456-4472, 4484-4505,4513-4516, 4531-4546, 4560-4650, 4664-4751, 4766-4918, 4932-5035,5049-5064, 5091-5189, 5203-5448, 5459-5503, 5508-5520, 5535-5654,5668-5863, 5877-6016, 6025-6029, 6054-6063, 6078-6215, 6229-6701,6715-6729, 6744-6869, 6883-6945, 6959-6968, 6982-7085, 7099-7173,7195-7247, 7255-7268, 7273-7309, 7320-7335, 7349-7442, 7456-7465,7479-7727, 7740-7951, 7977-8208, 8223-8255, 8257-8296, 8304-8312,8319-8329, 13093-13094SeqIDs w/o miR Seeds:30-32, 34-39, 45-62, 64-72, 77-142, 145-151, 153, 157-184, 186-202,205-246, 249, 251-260, 263, 266-320, 322, 326-328, 331, 333-341,343-344, 346-394, 396-445, 447-541, 543-562, 564-596, 599-605, 607-646,648-673, 677-701, 703-735, 737-772, 774-781, 785, 787-793, 795-809, 812,814-815, 819-820, 822-827, 833-834, 836, 838-850, 852-876, 879, 883-886,889-890, 892, 894, 897, 899, 901-906, 909, 911, 919, 921-935, 940,942-1012, 1016-1063, 1065-1067, 1069-1095, 1097-1147, 1149-1166,1168-1190, 1193-1214, 1217, 1227-1237, 1240, 1244, 1246-1251, 1258-1281,1283-1312, 1314-1333, 1335, 1337-1356, 1358-1364, 1367, 1369-1381, 1384,1388-1389, 1392-1404, 1406-1417, 1421-1441, 1443, 1445, 1447-1460,1462-1492, 1494-1500, 1502, 1506-1512, 1514-1531, 1533, 1535-1539,1543-1558, 1561-1562, 1564-1601, 1603-1614, 1616-1633, 1635-1646,1648-1656, 1658, 1661-1675, 1678-1716, 1718-1740, 1742-1750, 1752-1785,1788-1795, 1798, 1804-1871, 1873-1884, 1892-1973, 1976-1992, 1998-2032,2034-2053, 2055-2077, 2079-2116, 2118-2135, 2138, 2142, 2149, 2151-2153,2155-2162, 2165-2174, 2178, 2181-2254, 2256-2268, 2271-2293, 2296-2298,2301-2312, 2314-2323, 2325-2427, 2429-2438, 2441, 2445, 2450-2476,2478-2489, 2492-2494, 2497-2513, 2515, 2521-2526, 2529-2545, 2547-2571,2573-2610, 2613-2620, 2622-2639, 2641, 2644, 2651-2743, 2745, 2747-2755,2760-2775, 2777-2825, 2828-2841, 2844-2861, 2864-2888, 2894-2954,2956-2988, 2991-3006, 3008-3043, 3046, 3048-3239, 3241-3253, 3256-3268,3270-3273, 3276-3320, 3322-3355, 3357-3404, 3406-3428, 3430-3488,3490-3491, 3493-3522, 3524-3552, 3554-3569, 3571-3650, 3653-3670,3672-3688, 3690-3717, 3719-3724, 3727-3736, 3743, 3746, 3749, 3751-3821,3823-3842, 3844, 3846, 3848, 3851, 3855, 3858, 3861-3863, 3866-3881,3883, 3887-3907, 3909-3917, 3919-3924, 3926-3942, 3944-3952, 3956-3970,3976-3988, 3991-3998, 4001-4008, 4010-4022, 4026, 4028-4039, 4041-4048,4051-4059, 4063-4070, 4072-4073, 4075, 4078, 4080-4104, 4106-4124, 4126,4129-4140, 4143-4153, 4156-4162, 4166-4171, 4174-4196, 4201-4241,4244-4252, 4254-4285, 4289-4318, 4320-4324, 4327, 4330-4331, 4333-4335,4337-4351, 4353-4414, 4417, 4419-4425, 4427-4433, 4435, 4438-4446,4449-4450, 4452-4462, 4470-4472, 4474-4510, 4512-4517, 4520-4546, 4548,4551-4590, 4592-4619, 4623-4696, 4699, 4701, 4703-4752, 4755-4877,4879-4949, 4951-5007, 5010, 5013-5046, 5049-5059, 5061-5063, 5066,5069-5078, 5080-5119, 5121-5131, 5134-5168, 5171-5189, 5191-5228,5230-5341, 5343-5405, 5407-5426, 5429-5437, 5439-5476, 5478-5491,5494-5516, 5518-5556, 5558-5572, 5574-5647, 5649, 5652-5653, 5657-5729,5731-5742, 5744-5769, 5771-5780, 5782-5866, 5868, 5870-5876, 5879-5881,5884-5899, 5902-5951, 5954-5993, 6000-6006, 6009-6016, 6019-6033,6035-6065, 6067-6073, 6080-6165, 6168-6249, 6252-6257, 6261-6282,6284-6289, 6291-6301, 6305-6367, 6369-6378, 6380-6398, 6401-6412, 6415,6417-6447, 6449-6456, 6458-6500, 6502-6563, 6565-6589, 6591-6612,6616-6660, 6663-6702, 6704-6748, 6753, 6758-6763, 6765, 6768-6807, 6810,6812-6872, 6874-6877, 6879-6913, 6915-6916, 6919, 6922, 6924-6926, 6928,6930-6936, 6940-6959, 6962, 6964-6990, 6992, 6996, 6998-6999, 7004-7038,7042-7085, 7087, 7089, 7092-7134, 7136-7140, 7142-7143, 7146-7150,7152-7157, 7159-7171, 7175, 7177-7178, 7180-7196, 7198-7220, 7223,7231-7237, 7239, 7242-7246, 7250-7273, 7275-7308, 7310-7312, 7314-7317,7319-7330, 7332-7400, 7402-7437, 7439, 7441-7466, 7470-7491, 7493, 7495,7497, 7499, 7502-7614, 7622-7628, 7631-7646, 7649-7651, 7655-7657,7661-7672, 7676, 7679-7721, 7723-7800, 7802-7803, 7805-7906, 7908,7910-7939, 7943-7953, 7956-7964, 7966-7981, 7983, 7985-7999, 8002,8004-8034, 8036-8046, 8048-8080, 8084-8094, 8096-8112, 8114-8115,8117-8139, 8141-8143, 8146-8148, 8150-8187, 8190-8216, 8218-8229,8232-8238, 8240-8250, 8253-8255, 8257-8275, 8278-8296, 8299-8304,8306-8329, 13093-13094Single Strand Oligonucleotides (Sense Strand of Target Gene)SeqID range: 1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471,2488-2490, 2542-2546, 2656-2657, 2833-2835, 3439-3440, 3916-3918,4469-4472, 4821, 5429, 5537, 6061, 7327, 8330-13061, 13062-13087SeqIDs w/o G Runs:1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2490, 2542-2545,2656-2657, 2833-2835, 3439-3440, 3916-3918, 4469-4472, 4821, 5429, 5537,6061, 7327, 8330-8495, 8520-8560, 8574-8837, 8857-8882, 8907-8964,8978-9298, 9312-9382, 9394-9640, 9656-9753, 9767-9974, 9988-10261,10275-10276, 10290-10301, 10315-10434, 10448-10613, 10623-10641,10644-10676, 10678-10704, 10714-10802, 10822-11161, 11175-11192,11207-11386, 11400-11730, 11744-11745, 11759-11852, 11857-11900,11914-11984, 11999-12011, 12026-12153, 12163-12175, 12178-12195,12198-12212, 12216-12536, 12547-12564, 12575-12664, 12674-12758,12772-12797, 12800-12840, 12854-13061, 13062-13069SeqIDs w/o miR Seeds:1158-1159, 1171, 1482-1483, 1485-1486, 2465-2471, 2488-2489, 2542-2545,2656-2657, 2833-2835, 3439-3440, 3916-3917, 4470-4472, 4821, 5429, 5537,6061, 7327, 8330-8334, 8336-8345, 8347, 8351-8373, 8375-8390, 8392-8399,8401-8413, 8415-8455, 8457-8493, 8495, 8497-8502, 8510-8517, 8520, 8525,8527-8634, 8637-8653, 8655-8671, 8673-8718, 8721-8822, 8824-8825,8827-8842, 8849-8879, 8881-8892, 8894-8902, 8905-8906, 8914-8927, 8929,8931, 8935, 8937-8975, 8980-8992, 8994, 8996-8997, 8999-9001, 9003,9005-9086, 9089-9124, 9126-9286, 9288-9307, 9310-9359, 9362-9420,9425-9427, 9429-9432, 9434, 9436-9437, 9439-9461, 9464-9483, 9486,9488-9498, 9500-9511, 9513, 9515-9650, 9653-9667, 9669, 9671-9723,9725-9869, 9871-9872, 9874-9879, 9881-9889, 9891-9973, 9975-10077,10080-10097, 10099, 10101-10127, 10129-10166, 10168-10170, 10172-10184,10186-10230, 10232-10237, 10239-10260, 10262-10272, 10274-10342,10344-10400, 10402-10423, 10426-10441, 10445-10556, 10560, 10562-10580,10582-10606, 10609-10647, 10650, 10652-10704, 10706, 10710-10713,10716-10731, 10733-10824, 10826-10842, 10844-10903, 10906-10907,10909-11101, 11104, 11106-11134, 11137-11138, 11145-11161, 11164-11173,11175-11181, 11184, 11186-11203, 11207-11212, 11214-11239, 11243-11259,11261-11347, 11351-11397, 11399-11740, 11742-11747, 11749-11790,11792-11817, 11821-11852, 11854-11904, 11908-11944, 11946-11959, 11961,11964-11984, 11986-12007, 12009-12022, 12024-12092, 12095-12119,12121-12133, 12135-12144, 12146-12157, 12159-12225, 12227-12231,12233-12300, 12302-12329, 12332-12333, 12335-12382, 12385-12411,12414-12416, 12418-12444, 12446-12455, 12457-12461, 12465, 12468-12474,12476-12499, 12501-12536, 12538-12544, 12547-12553, 12559-12610,12612-12626, 12628-12631, 12633-12637, 12640-12645, 12648-12657,12659-12671, 12673-12679, 12683-12710, 12712, 12714-12747, 12750,12752-12766, 12769, 12771-12806, 12808-12826, 12828-12829, 12831,12833-12846, 12848-12849, 12854-12931, 12933-12946, 12948-13061, 13064,13066-13068, 13071-13072, 13075, 13077-13081, 13083, 13085, 13087

Example 2: Selective Upregulation of Exon 7 Containing SMN2 TranscriptsUsing Oligonucleotides Targeting PRC2-Interacting Regions thatUpregulate SMN2 and Splice-Switching Oligonucleotides

Oligo Design:

Oligonucleotides targeting PRC2-interacting regions (lncRNA peaks) inthe SMN1/2 gene loci were designed. These oligos were synthesized withvarious DNA base modifications, modification placements,inter-nucleoside bonds and inter-oligo linkers (oligos 1-52 and 59-101)as outlined in Table 4.

Splice switching oligos (SSO) were designed based on sequences of SMN2.Various modifications of such SSOs in length and chemistry were prepared(oligos 53-58).

Universal negative control oligos (oligo 232 and 293) were also designedusing on bioinformatic analysis.

Methods:

Cell Culture:

Six SMA fibroblast cell lines and one lymphoblast cell line wereobtained from the Coriell Institute (FIG. 2). The cells were eithertransfected with the oligos using Lipofectamine 2000 (Fibroblasts) or byelectroporation or unassisted delivery (lymphoblast) to ascertaineffects of the oligonucleotides on SMN1/2 mRNA and protein expression.All experiments were carried out as biological triplicates.

mRNA and Protein Expression:

mRNA Expression—

1. On day 1, SMA fibroblasts were seeded into each well of 96-wellplates at a density of 5,000 cells per 100 uL.

2. On day 2, transfections were performed using Lipofectamie2000 permanufacturer's instructions with oligos at either 10 nM or 30 nM.

3. 48 hours post-transfection, Ambion Cells-to-CT kit was used todirectly obtain qRT-PCR results from the cells per manufacturer'sinstructions.

4. Quantitative PCR evaluation was completed using Taqman FAST qPCR onStepOne Plus, and change in mRNA expression was quantified using thedelta delta Ct method by normalizing SMN expression to a housekeepergene (B2M).

Protein Expression (ELISA)—

ELISA to determine SMN protein was carried out per manufacturer'sinstructions (SMN ELISA kit # ADI-900-209, Enzo Life Sciences). Briefly,SMN fibroblasts were cultures at 40,000 cells/well of a 24-well tissueculture coated plate on day 1. Cells were transfected with the oligosusing Lipofectamine2000 on day 2 and cell lysates prepared at 24 and 48hours post-treatment. ELISA was carried out per manufacturer'sinstructions. Subsequently fold induction of SMN protein was determinedby normalizing SMN protein levels induced by oligonucleotides to the SMNprotein levels induced by Lipofectamine treatment alone.

Splice Switching Assay (DdeI Assay)—

SMN2-derived transcripts contain a unique DdeI restriction elementintroduced because of a nucleotide polymorphism not present in SMN1 andare differentiated from SMN1-derived transcripts because of the fastermigration of the SMN2 products. Briefly, SMA fibroblasts were treatedwith oligonucleotides targeting PRC2-interacting regions with or withoutSSO at 30 nM each as described before. RT-PCR was carried out with anSMN exon 5 forward primer and an exon 8 reverse primer to generate cDNAsthat were then digested with DdeI. The SMN1 transcript, if present,migrates at a slower rate than the DdeI-digested SMN2 transcript and isseen as the first band from the top of the gel. The second band from thetop indicates full length SMN2 (accurately spliced form) and the thirdband indicates the incorrectly spliced SMN2delta7. (FIG. 5)

Results

In Spinal Muscular Atrophy patients, the SMN1 gene is often mutated insuch a way that it is unable to correctly code the SMN protein—due toeither a deletion encompassing at least a portion of exon 7 or to othermutations. SMA patients, however, generally retain at least one copy ofthe SMN2 gene (with many having 2 or more copies) that still expressessmall amounts of SMN protein. The SMN2 gene has a C to T mutation(compared with SMN1) in exon 7 that alters splicing of its precursormRNA such that exon 7 is spliced out at a high frequency. Consequently,only about 10% of the normal levels of full length SMN protein areproduced from SMN2. (See FIG. 1)

Six SMA fibroblast cell lines and one lymphoblast cell line wereobtained from the Coriell Institute (FIG. 2). Cells were transfectedwith oligonucleotides (oligos 1-52 and 59-101) directed against aPRC2-associated region of SMN2 and RT-PCR assays were conducted toevaluate effects on SMN mRNA expression. (See FIGS. 3 and 4 for resultsin cell lines 3814 and 3813). In separate experiments, cells weretransfected with oligonucleotides (oligos 53-58) directed at a splicecontrol sequence in intron 7 of SMN2 and RT-PCR assays were conducted toevaluate effects on SMN mRNA expression (See FIGS. 3 and 4 for resultsin cell lines 3814 and 3813). Splice switching oligonucleotides (oligos53-58) were found to increase expression of full length SMN2 based on agel separation analysis of PCR products obtained following a DdeIrestriction digest; whereas certain oligonucleotides directed against aPRC2-associated region of SMN2 did not. (FIG. 5)

SMN ELISA (Enzo) assays were conducted and revealed that certainoligonucleotides directed against a PRC2-associated region of SMN2 alonedid not significantly increase full length SMN protein 24 hpost-transfection in certain SMA patient fibroblasts. (FIGS. 6A-6B)However, the same SMN ELISA assays showed that oligonucleotides directedagainst a PRC2-associated region of SMN2 in combination with a spliceswitching oligonucleotide (oligo 53 or 54) significantly increase fulllength SMN protein 24 h post-transfection in SMA patient fibroblastsabove that observed with splice switching oligonucleotides alone. (FIGS.7A-B and 8A-8B). RT-PCR assays were conducted and showed thatoligonucleotides directed against a PRC2-associated region of SMN2 incombination with a splice switching oligonucleotide (oligo 53 or 54)significantly increased SMN2 protein 24 h post-transfection in SMApatient fibroblasts. (FIG. 9.) These experiments were conducted modifiedoligonucleotides with either alternating LNA and 2′OMe nucleotides oralternating DNA and LNA nucleotides.

In summary, the results of Example 2 show that certain oligos targetingPRC2 associated regions of SMN2 induce SMN RNA expression (e.g., of theSMNA7 transcript) SMA patient-derived fibroblasts. The results also showthat, in some embodiments, splice-switching oligos may not induce SMNRNA expression, but rather shift SMN RNA splicing to the full-lengthtranscript. Finally, the results show that combination of spliceswitching oligos with PRC2-associated region targeting oligossubstantially increases full length SMN protein in cells from SMApatients.

TABLE 4 Oligonucleotide sequences made for testing human cellsobtained from subjects with Spinal Muscular Atrophy(See Table 3 for structural features of formatted sequence). Oligo BaseName Sequence Formatted Sequence SeqID SMN1- ATTCTCTTGAomeAs;omeUs;omeUs;omeCs;omeUs;omeCs;omeUs;omeUs;omeGs; 13062 01 m03TGATGCTGAT omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeU-Sup SMN1- TTCTCTTGATomeUs;omeUs;omeCs;omeUs;omeCs;omeUs;omeUs;omeGs;omeAs; 13063 02 m03GATGCTGATG omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeG-Sup SMN1- TCTCTTGATGomeUs;omeCs;omeUs;omeCs;omeUs;omeUs;omeGs;omeAs;omeUs; 13064 03 m03ATGCTGATGC omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeC-Sup SMN1- CTCTTGATGAomeCs;omeUs;omeCs;omeUs;omeUs;omeGs;omeAs;omeUs;omeGs; 13065 04 m03TGCTGATGCT omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeU-Sup SMN1- TCTTGATGATomeUs;omeCs;omeUs;omeUs;omeGs;omeAs;omeUs;omeGs;omeAs; 13066 05 m03GCTGATGCTT omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeU-Sup SMN1- CTTGATGATGomeCs;omeUs;omeUs;omeGs;omeAs;omeUs;omeGs;omeAs;omeUs; 13067 06 m03CTGATGCTTT omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeU-Sup SMN1- TTGATGATGComeUs;omeUs;omeGs;omeAs;omeUs;omeGs;omeAs;omeUs;omeGs; 13068 07 m03TGATGCTTTG omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeG-Sup SMN1- TGATGATGCTomeUs;omeGs;omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs; 13069 08 m03GATGCTTTGG omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeG-Sup SMN1- GATGATGCTGomeGs;omeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs; 13070 09 m03ATGCTTTGGG omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeG-Sup SMN1- ATGATGCTGAomeAs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs; 13071 10 m03TGCTTTGGGA omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeA-Sup SMN1- TGATGCTGATomeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs; 13072 11 m03GCTTTGGGAA omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeA-Sup SMN1- GATGCTGATGomeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs; 13073 12 m03CTTTGGGAAG omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeG-Sup SMN1- ATGCTGATGComeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs; 13074 13 m03TTTGGGAAGT omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeU-Sup SMN1- TGCTGATGCTomeUs;omeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs; 13075 14 m03TTGGGAAGTA omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeA-Sup SMN1- GCTGATGCTTomeGs;omeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs; 13076 15 m03TGGGAAGTAT omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeU-Sup SMN1- CTGATGCTTTomeCs;omeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs; 13077 16 m03GGGAAGTATG omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeG-Sup SMN1- TGATGCTTTGomeUs;omeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs; 13078 17 m03GGAAGTATGT omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeU-Sup SMN1- GATGCTTTGGomeGs;omeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs; 13079 18 m03GAAGTATGTT omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeU-Sup SMN1- ATGCTTTGGGomeAs;omeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs; 13080 19 m03AAGTATGTTA omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeA-Sup SMN1- TGCTTTGGGAomeUs;omeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs; 13081 20 m03AGTATGTTAA omeAs;omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeA-Sup SMN1- GCTTTGGGAAomeGs;omeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs; 13082 21 m03GTATGTTAAT omeAs;omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeU-Sup SMN1- CTTTGGGAAGomeCs;omeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs; 13083 22 m03TATGTTAATT omeGs;omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeU-Sup SMN1- TTTGGGAAGTomeUs;omeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs; 13084 23 m03ATGTTAATTT omeUs;omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeU-Sup SMN1- TTGGGAAGTAomeUs;omeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs; 13085 24 m03TGTTAATTTC omeAs;omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeUs;omeC-Sup SMN1- TGGGAAGTATomeUs;omeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs; 13086 25 m03GTTAATTTCA omeUs;omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeUs;omeCs;omeA-Sup SMN1- GGGAAGTATGomeGs;omeGs;omeGs;omeAs;omeAs;omeGs;omeUs;omeAs;omeUs; 13087 26 m03TTAATTTCAT omeGs;omeUs;omeUs;omeAs;omeAs;omeUs;omeUs;omeUs;omeCs;omeAs;omeU-Sup SMN1- ATTCTCTTGAInaAs;omeUs;InaTs;omeCs;InaTs;omeCs;InaTs;omeUs;InaGs; 11374 27 m01TGATG omeAs;InaTs;omeGs;InaAs;omeUs;InaG-Sup SMN1- TTCTCTTGATInaTs;omeUs;InaCs;omeUs;InaCs;omeUs;InaTs;omeGs;InaAs; 11375 28 m01GATGC omeUs;InaGs;omeAs;InaTs;omeGs;InaC-Sup SMN1- TCTCTTGATGInaTs;omeCs;InaTs;omeCs;InaTs;omeUs;InaGs;omeAs;InaTs; 11376 29 m01ATGCT omeGs;InaAs;omeUs;InaGs;omeCs;InaT-Sup SMN1- CTCTTGATGAInaCs;omeUs;InaCs;omeUs;InaTs;omeGs;InaAs;omeUs;InaGs; 11377 30 m01TGCTG omeAs;InaTs;omeGs;InaCs;omeUs;InaG-Sup SMN1- TCTTGATGATInaTs;omeCs;InaTs;omeUs;InaGs;omeAs;InaTs;omeGs;InaAs; 11378 31 m01GCTGA omeUs;InaGs;omeCs;InaTs;omeGs;InaA-Sup SMN1- CTTGATGATGInaCs;omeUs;InaTs;omeGs;InaAs;omeUs;InaGs;omeAs;InaTs; 11379 32 m01CTGAT omeGs;InaCs;omeUs;InaGs;omeAs;InaT-Sup SMN1- TTGATGATGCInaTs;omeUs;InaGs;omeAs;InaTs;omeGs;InaAs;omeUs;InaGs; 11380 33 m01TGATG omeCs;InaTs;omeGs;InaAs;omeUs;InaG-Sup SMN1- TGATGATGCTInaTs;omeGs;InaAs;omeUs;InaGs;omeAs;InaTs;omeGs;InaCs; 11381 34 m01GATGC omeUs;InaGs;omeAs;InaTs;omeGs;InaC-Sup SMN1- GATGATGCTGInaGs;omeAs;InaTs;omeGs;InaAs;omeUs;InaGs;omeCs;InaTs; 11382 35 m01ATGCT omeGs;InaAs;omeUs;InaGs;omeCs;InaT-Sup SMN1- ATGATGCTGAInaAs;omeUs;InaGs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs; 11383 36 m01TGCTT omeAs;InaTs;omeGs;InaCs;omeUs;InaT-Sup SMN1- TGATGCTGATInaTs;omeGs;InaAs;omeUs;InaGs;omeCs;InaTs;omeGs;InaAs; 11384 37 m01GCTTT omeUs;InaGs;omeCs;InaTs;omeUs;InaT-Sup SMN1- GATGCTGATGInaGs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeAs;InaTs; 11385 38 m01CTTTG omeGs;InaCs;omeUs;InaTs;omeUs;InaG-Sup SMN1- ATGCTGATGCInaAs;omeUs;InaGs;omeCs;InaTs;omeGs;InaAs;omeUs;InaGs; 11386 39 m01TTTGG omeCs;InaTs;omeUs;InaTs;omeGs;InaG-Sup SMN1- TGCTGATGCTInaTs;omeGs;InaCs;omeUs;InaGs;omeAs;InaTs;omeGs;InaCs; 11387 40 m01TTGGG omeUs;InaTs;omeUs;InaGs;omeGs;InaG-Sup SMN1- GCTGATGCTTInaGs;omeCs;InaTs;omeGs;InaAs;omeUs;InaGs;omeCs;InaTs; 11388 41 m01TGGGA omeUs;InaTs;omeGs;InaGs;omeGs;InaA-Sup SMN1- CTGATGCTTTInaCs;omeUs;InaGs;omeAs;InaTs;omeGs;InaCs;omeUs;InaTs; 11389 42 m01GGGAA omeUs;InaGs;omeGs;InaGs;omeAs;InaA-Sup SMN1- TGATGCTTTGInaTs;omeGs;InaAs;omeUs;InaGs;omeCs;InaTs;omeUs;InaTs; 11390 43 m01GGAAG omeGs;InaGs;omeGs;InaAs;omeAs;InaG-Sup SMN1- GATGCTTTGGInaGs;omeAs;InaTs;omeGs;InaCs;omeUs;InaTs;omeUs;InaGs; 11391 44 m01GAAGT omeGs;InaGs;omeAs;InaAs;omeGs;InaT-Sup SMN1- ATGCTTTGGGInaAs;omeUs;InaGs;omeCs;InaTs;omeUs;InaTs;omeGs;InaGs; 11392 45 m01AAGTA omeGs;InaAs;omeAs;InaGs;omeUs;InaA-Sup SMN1- TGCTTTGGGAInaTs;omeGs;InaCs;omeUs;InaTs;omeUs;InaGs;omeGs;InaGs; 11393 46 m01AGTAT omeAs;InaAs;omeGs;InaTs;omeAs;InaT-Sup SMN1- GCTTTGGGAAdGs;InaCs;dTs;InaTs;dTs;InaGs;dGs;InaGs;dAs;InaAs;dGs; 11394 47 m02GTATG InaTs;dAs;InaTs;dG-Sup SMN1- GCTTTGGGAAInaGs;omeCs;InaTs;omeUs;InaTs;omeGs;InaGs;omeGs;InaAs; 11394 47 m01GTATG omeAs;InaGs;omeUs;InaAs;omeUs;InaG-Sup SMN1- CTTTGGGAAGdCs;InaTs;dTs;InaTs;dGs;InaGs;dGs;InaAs;dAs;InaGs;dTs; 11395 48 m05TATGT InaAs;dTs;InaGs;dT-Sup SMN1- CTTTGGGAAGInaCs;omeUs;InaTs;omeUs;InaGs;omeGs;InaGs;omeAs;InaAs; 11395 48 m01TATGT omeGs;InaTs;omeAs;InaTs;omeGs;InaT-Sup SMN1- TTTGGGAAGTInaTs;omeUs;InaTs;omeGs;InaGs;omeGs;InaAs;omeAs;InaGs; 11396 49 m01ATGTT omeUs;InaAs;omeUs;InaGs;omeUs;InaT-Sup SMN1- TTGGGAAGTAInaTs;omeUs;InaGs;omeGs;InaGs;omeAs;InaAs;omeGs;InaTs; 11397 50 m01TGTTA omeAs;InaTs;omeGs;InaTs;omeUs;InaA-Sup SMN1- TGGGAAGTATInaTs;omeGs;InaGs;omeGs;InaAs;omeAs;InaGs;omeUs;InaAs; 11398 51 m01GTTAA omeUs;InaGs;omeUs;InaTs;omeAs;InaA-Sup SMN1- GGGAAGTATGInaGs;omeGs;InaGs;omeAs;InaAs;omeGs;InaTs;omeAs;InaTs; 11399 52 m01TTAAT omeGs;InaTs;omeUs;InaAs;omeAs;InaT-Sup SMN1- TCACTTTCATdTs;InaCs;dAs;InaCs;dTs;InaTs;dTs;InaCs;dAs;InaTs;dAs; 13088 53 m02AATGCTGG InaAs;dTs;InaGs;dCs;InaTs;dGs;InaG-Sup SMN1- TCACTTTCATInaTs;dCs;InaAs;dCs;InaTs;dTs;InaTs;dCs;InaAs;dTs; 13088 53 m12 AATGCTGGInaAs;dAs;InaTs;dGs;InaCs;dTs;InaGs;dG-Sup SMN1- TCACTTTCATInaTs;omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs; 13088 54 m01AATGCTGG omeUs;InaAs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeG- Sup SMN1-TCACTTTCAT omeUs;omeCs;omeAs;omeCs;omeUs;omeUs;omeUs;omeCs;omeAs; 1308853 m03 AATGCTGG omeUs;omeAs;omeAs;omeUs;omeGs;omeCs;omeUs;omeGs;omeG-Sup SMN1- TCACTTTCATInaTs;omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs; 13089 55 m01AATGC omeUs;InaAs;omeAs;InaTs;omeGs;InaC-Sup SMN1- CACTTTCATAInaCs;omeAs;InaCs;omeUs;InaTs;omeUs;InaCs;omeAs;InaTs; 13090 56 m01ATGCT omeAs;InaAs;omeUs;InaGs;omeCs;InaT-Sup SMN1- ACTTTCATAAdAs;InaCs;dTs;InaTs;dTs;InaCs;dAs;InaTs;dAs;InaAs;dTs; 13091 57 m02TGCTG InaGs;dCs;InaTs;dG-Sup SMN1- ACTTTCATAAInaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs;omeUs;InaAs; 13091 57 m01TGCTG omeAs;InaTs;omeGs;InaCs;omeUs;InaG-Sup SMN1- CTTTCATAATInaCs;omeUs;InaTs;omeUs;InaCs;omeAs;InaTs;omeAs;InaAs; 13092 58 m01GCTGG omeUs;InaGs;omeCs;InaTs;omeGs;InaG-Sup SMN1- AGACCAGTTTInaAs;omeGs;InaAs;omeCs;InaCs;omeAs;InaGs;omeUs;InaTs;  3650 59 m01TACCT omeUs;InaTs;omeAs;InaCs;omeCs;InaT-Sup SMN1- CCTAGCTACTInaCs;omeCs;InaTs;omeAs;InaGs;omeCs;InaTs;omeAs;InaCs; 13093 60 m01TTGAA omeUs;InaTs;omeUs;InaGs;omeAs;InaA-Sup SMN1- TCCTAGCTACInaTs;omeCs;InaCs;omeUs;InaAs;omeGs;InaCs;omeUs;InaAs; 13094 61 m01TTTGA omeCs;InaTs;omeUs;InaTs;omeGs;InaA-Sup SMN1- GAAATATTCCInaGs;omeAs;InaAs;omeAs;InaTs;omeAs;InaTs;omeUs;InaCs; 10065 62 m01TTATA omeCs;InaTs;omeUs;InaAs;omeUs;InaA-Sup SMN1- AAATATTCCTInaAs;omeAs;InaAs;omeUs;InaAs;omeUs;InaTs;omeCs;InaCs; 10066 63 m01TATAG omeUs;InaTs;omeAs;InaTs;omeAs;InaG-Sup SMN1- AATATTCCTTInaAs;omeAs;InaTsiomeAs;InaTs;omeUs;InaCs;omeCs;InaTs; 10067 64 m01ATAGC omeUs;InaAs;omeUs;InaAs;omeGs;InaC-Sup SMN1- ATATTCCTTAInaAs;omeUs;InaAs;omeUs;InaTs;omeCs;InaCs;omeUs;InaTs; 10068 65 m01TAGCC omeAs;InaTs;omeAs;InaGs;omeCs;InaC-Sup SMN1- TATTCCTTATInaTs;omeAs;InaTs;omeUs;InaCs;omeCs;InaTs;omeUs;InaAs; 10069 66 m01AGCCA omeUs;InaAs;omeGs;InaCs;omeCs;InaA-Sup SMN1- ATTCCTTATAInaAs;omeUs;InaTs;omeCs;InaCs;omeUs;InaTs;omeAs;InaTs; 10070 67 m01GCCAG omeAs;InaGs;omeCs;InaCs;omeAs;InaG-Sup SMN1- TTCCTTATAGInaTs;omeUs;InaCs;omeCs;InaTs;omeUs;InaAs;omeUs;InaAs; 10071 68 m01CCAGG omeGs;InaCs;omeCs;InaAs;omeGs;InaG-Sup SMN1- TCCTTATAGCInaTs;omeCs;InaCs;omeUs;InaTs;omeAs;InaTs;omeAs;InaGs; 10072 69 m01CAGGT omeCs;InaCs;omeAs;InaGs;omeGs;InaT-Sup SMN1- CCTTATAGCCInaCs;omeCs;InaTs;omeUs;InaAs;omeUs;InaAs;omeGs;InaCs; 10073 70 m01AGGTC omeCs;InaAs;omeGs;InaGs;omeUs;InaC-Sup SMN1- CTTATAGCCAInaCs;omeUs;InaTs;omeAs;InaTs;omeAs;InaGs;omeCs;InaCs; 10074 71 m01GGTCT omeAs;InaGs;omeGs;InaTs;omeCs;InaT-Sup SMN1- TTATAGCCAGInaTs;omeUs;InaAs;omeUs;InaAs;omeGs;InaCs;omeCs;InaAs; 10075 72 m01GTCTA omeGs;InaGs;omeUs;InaCs;omeUs;InaA-Sup SMN1- GCCAGGTCTAInaGs;omeCs;InaCs;omeAs;InaGs;omeGs;InaTs;omeCs;InaTs; 10080 73 m01AAATT omeAs;InaAs;omeAs;InaAs;omeUs;InaT-Sup SMN1- CCAGGTCTAAInaCs;omeCs;InaAs;omeGs;InaGs;omeUs;InaCs;omeUs;InaAs; 10081 74 m01AATTC omeAs;InaAs;omeAs;InaTs;omeUs;InaC-Sup SMN1- CAGGTCTAAAInaCs;omeAs;InaGs;omeGs;InaTs;omeCs;InaTs;omeAs;InaAs; 10082 75 m01ATTCA omeAs;InaAs;omeUs;InaTs;omeCs;InaA-Sup SMN1- GGTCTAAAATInaGs;omeGs;InaTs;omeCs;InaTs;omeAs;InaAs;omeAs;InaAs; 10084 76 m01TCAAT omeUs;InaTs;omeCs;InaAs;omeAs;InaT-Sup SMN1- CTAAAATTCAInaCs;omeUs;InaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs; 10087 77 m01ATGGC omeAs;InaAs;omeUs;InaGs;omeGs;InaC-Sup SMN1- CTAAAATTCAomeCs;omeUs;omeAs;omeAs;omeAs;omeAs;omeUs;omeUs;omeCs; 10087 77 m03ATGGC omeAs;omeAs;omeUs;omeGs;omeGs;omeC-Sup SMN1- GGACCACCAGInaGs;omeGs;InaAs;omeCs;InaCs;omeAs;InaCs;omeCs;InaAs; 10168 78 m01TAAGT omeGs;InaTs;omeAs;InaAs;omeGs;InaT-Sup SMN1- GACCACCAGTdGs;InaAs;dCs;InaCs;dAs;InaCs;dCs;InaAs;dGs;InaTs;dAs; 10169 79 m02AAGTA InaAs;dGs;InaTs;dA-Sup SMN1- GACCACCAGTInaGs;omeAs;InaCs;omeCs;InaAs;omeCs;InaCs;omeAs;InaGs; 10169 79 m01AAGTA omeUs;InaAs;omeAs;InaGs;omeUs;InaA-Sup SMN1- ACCACCAGTAdAs;InaCs;dCs;InaAs;dCs;InaCs;dAs;InaGs;dTs;InaAs;dAs; 10170 80 m02AGTAA InaGs;dTs;InaAs;dA-Sup SMN1- ACCACCAGTAInaAs;omeCs;InaCs;omeAs;InaCs;omeCs;InaAs;omeGs;InaTs; 10170 80 m01AGTAA omeAs;InaAs;omeGs;InaTs;omeAs;InaA-Sup SMN1- TTCTGTTACCInaTs;omeUs;InaCs;omeUs;InaGs;omeUs;InaTs;omeAs;InaCs; 10337 81 m01CAGAT omeCs;InaCs;omeAs;InaGs;omeAs;InaT-Sup SMN1- TCTGTTACCCInaTs;omeCs;InaTs;omeGs;InaTs;omeUs;InaAs;omeCs;InaCs; 10338 82 m01AGATG omeCs;InaAs;omeGs;InaAs;omeUs;InaG-Sup SMN1- CTGTTACCCAInaCs;omeUs;InaGs;omeUs;InaTs;omeAs;InaCs;omeCs;InaCs; 10339 83 m01GATGC omeAs;InaGs;omeAs;InaTs;omeGs;InaC-Sup SMN1- TTTTTAGGTAdTs;InaTs;dTs;InaTs;dTs;InaAs;dGs;InaGs;dTs;InaAs;dTs; 10763 84 m02TTAAC InaTs;dAs;InaAs;dC-Sup SMN1- TTTTTAGGTAInaTs;omeUs;InaTs;omeUs;InaTs;omeAs;InaGs;omeGs;InaTs; 10763 84 m01TTAAC omeAs;InaTs;omeUs;InaAs;omeAs;InaC-Sup SMN1- TTTTAGGTATInaTs;omeUs;InaTs;omeUs;InaAs;omeGs;InaGs;omeUs;InaAs; 10764 85 m01TAACA omeUs;InaTs;omeAs;InaAs;omeCs;InaA-Sup SMN1- CATAGCTTCAInaCs;omeAs;InaTs;omeAs;InaGs;omeCs;InaTs;omeUs;InaCs; 10949 86 m01TAGTG omeAs;InaTs;omeAs;InaGs;omeUs;InaG-Sup SMN1- TAGCTTCATAInaTs;omeAs;InaGs;omeCs;InaTs;omeUs;InaCs;omeAs;InaTs; 10951 87 m01GTGGA omeAs;InaGs;omeUs;InaGs;omeGs;InaA-Sup SMN1- AGCTTCATAGInaAs;omeGs;InaCs;omeUs;InaTs;omeCs;InaAs;omeUs;InaAs; 10952 88 m01TGGAA omeGs;InaTs;omeGs;InaGs;omeAs;InaA-Sup SMN1- GCTTCATAGTInaGs;omeCs;InaTs;omeUs;InaCs;omeAs;InaTs;omeAs;InaGs; 10953 89 m01GGAAC omeUs;InaGs;omeGs;InaAs;omeAs;InaC-Sup SMN1- CTTCATAGTGInaCs;omeUs;InaTs;omeCs;InaAs;omeUs;InaAs;omeGs;InaTs; 10954 90 m01GAACA omeGs;InaGs;omeAs;InaAs;omeCs;InaA-Sup SMN1- TCATGGTACAInaTs;omeCs;InaAs;omeUs;InaGs;omeGs;InaTs;omeAs;InaCs; 11415 91 m01TGAGT omeAs;InaTs;omeGs;InaAs;omeGs;InaT-Sup SMN1- TGGTACATGAInaTs;omeGs;InaGs;omeUs;InaAs;omeCs;InaAs;omeUs;InaGs; 11418 92 m01GTGGC omeAs;InaGs;omeUs;InaGs;omeGs;InaC-Sup SMN1- GGTACATGAGdGs;InaGs;dTs;InaAs;dCs;InaAs;dTs;InaGs;dAs;InaGs;dTs; 11419 93 m02TGGCT InaGs;dGs;InaCs;dT-Sup SMN1- GGTACATGAGInaGs;omeGs;InaTs;omeAs;InaCs;omeAs;InaTs;omeGs;InaAs; 11419 93 m01TGGCT omeGs;InaTs;omeGs;InaGs;omeCs;InaT-Sup SMN1- TACATGAGTGInaTs;omeAs;InaCs;omeAs;InaTs;omeGs;InaAs;omeGs;InaTs; 11421 94 m01GCTAT omeGs;InaGs;omeCs;InaTs;omeAs;InaT-Sup SMN1- ACATGAGTGGInaAs;omeCs;InaAs;omeUs;InaGs;omeAs;InaGs;omeUs;InaGs; 11422 95 m01CTATC omeGs;InaCs;omeUs;InaAs;omeUs;InaC-Sup SMN1- CATGAGTGGCInaCs;omeAs;InaTs;omeGs;InaAs;omeGs;InaTs;omeGs;InaGs; 11423 96 m01TATCA omeCs;InaTs;omeAs;InaTs;omeCs;InaA-Sup SMN1- CTGGCTATTAInaCs;omeUs;InaGs;omeGs;InaCs;omeUs;InaAs;omeUs;InaTs; 11440 97 m01TATGG omeAs;InaTs;omeAs;InaTs;omeGs;InaG-Sup SMN1- TGGCTATTATInaTs;omeGs;InaGs;omeCs;InaTs;omeAs;InaTs;omeUs;InaAs; 11441 98 m01ATGGT omeUs;InaAs;omeUs;InaGs;omeGs;InaT-Sup SMN1- GGCTATTATAInaGs;omeGs;InaCs;omeUs;InaAs;omeUs;InaTs;omeAs;InaTs; 11442 99 m01TGGTA omeAs;InaTs;omeGs;InaGs;omeUs;InaA-Sup SMN1- GCTATTATATInaGs;omeCs;InaTs;omeAs;InaTs;omeUs;InaAs;omeUs;InaAs; 11443 100 m01GGTAA omeUs;InaGs;omeGs;InaTs;omeAs;InaA-Sup SMN1- GTATCATCTGInaGs;omeUs;InaAs;omeUs;InaCs;omeAs;InaTs;omeCs;InaTs; 12369 101 m01TGTGT omeGs;InaTs;omeGs;InaTs;omeGs;InaT-Sup SMN1- GCTTTGGGAAInaGs;omeCs;InaTs;omeUs;InaTs;omeGs;InaGs;omeGs;InaAs; 13097 102GTATGTTTTT omeAs;InaGs;omeUs;InaAs;omeUs;InaG;dT;dT;dT;dT;InaTs; m01CACTTTCATA omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs;omeUs;ATGCTGG InaAs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeG-Sup SMN1-CTTTGGGAAG InaCs;omeUs;InaTs;omeUs;InaGs;omeGs;InaGs;omeAs;InaAs; 13102103 TATGTTTTTT omeGs;InaTs;omeAs;InaTs;omeGs;InaT;dT;dT;dT;dT;InaTs; m01CACTTTCATA omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs;omeUs;ATGCTGG InaAs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeG-Sup SMN1-GGTACATGAG InaGs;omeGs;InaTs;omeAs;InaCs;omeAs;InaTs;omeGs;InaAs; 13099104 TGGCTTTTTT omeGs;InaTs;omeGs;InaGs;omeCs;InaT;dT;dT;dT;dT;InaTs; m01CACTTTCATA omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs;omeUs;ATGCTGG InaAs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeG-Sup SMN1-TGATGCTGAT InaTs;omeGs;InaAs;omeUs;InaGs;omeCs;InaTs;omeGs;InaAs; 13103105 GCTTTTTTTC omeUs;InaGs;omeCs;InaTs;omeUs;InaT;dT;dT;dT;dT;InaCs; m01TAAAATTCAA omeUs;InaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs;omeAs; TGGCInaAs;omeUs;InaGs;omeGs;InaC-Sup SMN1- CTAAAATTCAInaCs;omeUs;InaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs; 13104 106ATGGCTTTTC omeAs;InaAs;omeUs;InaGs;omeGs;InaC;dT;dT;dT;dT;InaCs; m01TAAAATTCAA omeUs;InaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs;omeAs; TGGCInaAs;omeUs;InaGs;omeGs;InaC-Sup SMN1- CTGTTACCCAInaCs;omeUs;InaGs;omeUs;InaTs;omeAs;InaCs;omeCs;InaCs; 13105 107GATGCTTTTC omeAs;InaGs;omeAs;InaTs;omeGs;InaC;dT;dT;dT;dT;InaCs; m01TAAAATTCAA omeUs;InaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs;omeAs; TGGCInaAs;omeUs;InaGs;omeGs;InaC-Sup SMN1- CTTCATAGTGInaCs;omeUs;InaTs;omeCs;InaAs;omeUs;InaAs;omeGs;InaTs; 13106 108GAACATTTTC omeGs;InaGs;omeAs;InaAs;omeCs;InaA;dT;dT;dT;dT;InaCs; m01TAAAATTCAA omeUs;InaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs;omeAs; TGGCInaAs;omeUs;InaGs;omeGs;InaC-Sup SMN1- TCACTTTCATInaTs;omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;omeCs;InaAs; 13107 109AATGCTGGTT omeUs;InaAs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeG; m01TTTCACTTTC dT;dT;dT;dT;InaTs;omeCs;InaAs;omeCs;InaTs;omeUs;InaTs;ATAATGCTGG omeCs;InaAs;omeUs;InaAs;omeAs;InaTs;omeGs;InaCs;omeUs;InaGs;omeG-Sup SMN1- AAATTCAATGInaAs;omeAs;InaAs;omeUs;InaTs;omeCs;InaAs;omeAs;InaTs; 10090 110 m01GCCCA omeGs;InaGs;omeCs;InaCs;omeCs;InaA-Sup SMN1- AATTCAATGGInaAs;omeAs;InaTs;omeUs;InaCs;omeAs;InaAs;omeUs;InaGs; 10091 111 m01CCCAC omeGs;InaCs;omeCs;InaCs;omeAs;InaC-Sup SMN1- ATTCAATGGCInaAs;omeUs;InaTs;omeCs;InaAs;omeAs;InaTs;omeGs;InaGs; 10092 112 m01CCACC omeCs;InaCs;omeCs;InaAs;omeCs;InaC-Sup SMN1- TTCAATGGCCInaTs;omeUs;InaCs;omeAs;InaAs;omeUs;InaGs;omeGs;InaCs; 10093 113 m01CACCA omeCs;InaCs;omeAs;InaCs;omeCs;InaA-Sup SMN1- TCAATGGCCCInaTs;omeCs;InaAs;omeAs;InaTs;omeGs;InaGs;omeCs;InaCs; 10094 114 m01ACCAC omeCs;InaAs;omeCs;InaCs;omeAs;InaC-Sup SMN1- CAATGGCCCAInaCs;omeAs;InaAs;omeUs;InaGs;omeGs;InaCs;omeCs;InaCs; 10095 115 m01CCACC omeAs;InaCs;omeCs;InaAs;omeCs;InaC-Sup SMN1- AATGGCCCACInaAs;omeAs;InaTs;omeGs;InaGs;omeCs;InaCs;omeCs;InaAs; 10096 116 m01CACCG omeCs;InaCs;omeAs;InaCs;omeCs;InaG-Sup SMN1- ATGGCCCACCInaAs;omeUs;InaGs;omeGs;InaCs;omeCs;InaCs;omeAs;InaCs; 10097 117 m01ACCGC omeCs;InaAs;omeCs;InaCs;omeGs;InaC-Sup SMN1- AATGCCTTTCInaAs;omeAs;InaTs;omeGs;InaCs;omeCs;InaTs;omeUs;InaTs; 10330 118 m01TGTTA omeCs;InaTs;omeGs;InaTs;omeUs;InaA-Sup SMN1- ATGCCTTTCTInaAs;omeUs;InaGs;omeCs;InaCs;omeUs;InaTs;omeUs;InaCs; 10331 119 m01GTTAC omeUs;InaGs;omeUs;InaTs;omeAs;InaC-Sup SMN1- TGCCTTTCTGInaTs;omeGs;InaCs;omeCs;InaTs;omeUs;InaTs;omeCs;InaTs; 10332 120 m01TTACC omeGs;InaTs;omeUs;InaAs;omeCs;InaC-Sup SMN1- GCCTTTCTGTInaGs;omeCs;InaCs;omeUs;InaTs;omeUs;InaCs;omeUs;InaGs; 10333 121 m01TACCC omeUs;InaTs;omeAs;InaCs;omeCs;InaC-Sup SMN1- CCTTTCTGTTInaCs;omeCs;InaTs;omeUs;InaTs;omeCs;InaTs;omeGs;InaTs; 10334 122 m01ACCCA omeUs;InaAs;omeCs;InaCs;omeCs;InaA-Sup SMN1- CTTTCTGTTAInaCs;omeUs;InaTs;omeUs;InaCs;omeUs;InaGs;omeUs;InaTs; 10335 123 m01CCCAG omeAs;InaCs;omeCs;InaCs;omeAs;InaG-Sup SMN1- TTTCTGTTACInaTs;omeUs;InaTs;omeCs;InaTs;omeGs;InaTs;omeUs;InaAs; 10336 124 m01CCAGA omeCs;InaCs;omeCs;InaAs;omeGs;InaA-Sup SMN1- TGTTACCCAGInaTs;omeGs;InaTs;omeUs;InaAs;omeCs;InaCs;omeCs;InaAs; 10340 125 m01ATGCA omeGs;InaAs;omeUs;InaGs;omeCs;InaA-Sup SMN1- GTTACCCAGAInaGs;omeUs;InaTs;omeAs;InaCs;omeCs;InaCs;omeAs;InaGs; 10341 126 m01TGCAG omeAs;InaTs;omeGs;InaCs;omeAs;InaG-Sup SMN1- TTACCCAGATInaTs;omeUs;InaAs;omeCs;InaCs;omeCs;InaAs;omeGs;InaAs; 10342 127 m01GCAGT omeUs;InaGs;omeCs;InaAs;omeGs;InaT-Sup SMN1- ACCCAGATGCInaAs;omeCs;InaCs;omeCs;InaAs;omeGs;InaAs;omeUs;InaGs; 10344 128 m01AGTGC omeCs;InaAs;omeGs;InaTs;omeGs;InaC-Sup SMN1- CCCAGATGCAInaCs;omeCs;InaCs;omeAs;InaGs;omeAs;InaTs;omeGs;InaCs; 10345 129 m01GTGCT omeAs;InaGs;omeUs;InaGs;omeCs;InaT-Sup SMN1- CCAGATGCAGInaCs;omeCs;InaAs;omeGs;InaAs;omeUs;InaGs;omeCs;InaAs; 10346 130 m01TGCTC omeGs;InaTs;omeGs;InaCs;omeUs;InaC-Sup SMN1- CAGATGCAGTInaCs;omeAs;InaGs;omeAs;InaTs;omeGs;InaCs;omeAs;InaGs; 10347 131 m01GCTCT omeUs;InaGs;omeCs;InaTs;omeCs;InaT-Sup SMN1- AGATGCAGTGInaAs;omeGs;InaAs;omeUs;InaGs;omeCs;InaAs;omeGs;InaTs; 10348 132 m01CTCTT omeGs;InaCs;omeUs;InaCs;omeUs;InaT-Sup SMN1- TTTTACTCATInaTs;omeUs;InaTs;omeUs;InaAs;omeCs;InaTs;omeCs;InaAs; 10942 133 m01AGCTT omeUs;InaAs;omeGs;InaCs;omeUs;InaT-Sup SMN1- TTTACTCATAInaTs;omeUs;InaTs;omeAs;InaCs;omeUs;InaCs;omeAs;InaTs; 10943 134 m01GCTTC omeAs;InaGs;omeCs;InaTs;omeUs;InaC-Sup SMN1- TTACTCATAGInaTs;omeUs;InaAs;omeCs;InaTs;omeCs;InaAs;omeUs;InaAs; 10944 135 m01CTTCA omeGs;InaCs;omeUs;InaTs;omeCs;InaA-Sup SMN1- TACTCATAGCInaTs;omeAs;InaCs;omeUs;InaCs;omeAs;InaTs;omeAs;InaGs; 10945 136 m01TTCAT omeCs;InaTs;omeUs;InaCs;omeAs;InaT-Sup SMN1- ACTCATAGCTInaAs;omeCs;InaTs;omeCs;InaAs;omeUs;InaAs;omeGs;InaCs; 10946 137 m01TCATA omeUs;InaTs;omeCs;InaAs;omeUs;InaA-Sup SMN1- CTCATAGCTTInaCs;omeUs;InaCs;omeAs;InaTs;omeAs;InaGs;omeCs;InaTs; 10947 138 m01CATAG omeUs;InaCs;omeAs;InaTs;omeAs;InaG-Sup SMN1- TCATAGCTTCInaTs;omeCs;InaAs;omeUs;InaAs;omeGs;InaCs;omeUs;InaTs; 10948 139 m01ATAGT omeCs;InaAs;omeUs;InaAs;omeGs;InaT-Sup SMN1- ATAGCTTCATInaAs;omeUs;InaAs;omeGs;InaCs;omeUs;InaTs;omeCs;InaAs; 10950 140 m01AGTGG omeUs;InaAs;omeGs;InaTs;omeGs;InaG-Sup SMN1- TTCATAGTGGInaTs;omeUs;InaCs;omeAs;InaTs;omeAs;InaGs;omeUs;InaGs; 10955 141 m01AACAG omeGs;InaAs;omeAs;InaCs;omeAs;InaG-Sup SMN1- TCATAGTGGAInaTs;omeCs;InaAs;omeUs;InaAs;omeGs;InaTs;omeGs;InaGs; 10956 142 m01ACAGA omeAs;InaAs;omeCs;InaAs;omeGs;InaA-Sup SMN1- CATAGTGGAAInaCs;omeAs;InaTs;omeAs;InaGs;omeUs;InaGs;omeGs;InaAs; 10957 143 m01CAGAT omeAs;InaCs;omeAs;InaGs;omeAs;InaT-Sup SMN1- ATAGTGGAACInaAs;omeUs;InaAs;omeGs;InaTs;omeGs;InaGs;omeAs;InaAs; 10958 144 m01AGATA omeCs;InaAs;omeGs;InaAs;omeUs;InaA-Sup SMN1- TAGTGGAACAInaTs;omeAs;InaGs;omeUs;InaGs;omeGs;InaAs;omeAs;InaCs; 10959 145 m01GATAC omeAs;InaGs;omeAs;InaTs;omeAs;InaC-Sup SMN1- AGTGGAACAGInaAs;omeGs;InaTs;omeGs;InaGs;omeAs;InaAs;omeCs;InaAs; 10960 146 m01ATACA omeGs;InaAs;omeUs;InaAs;omeCs;InaA-Sup SMN1- GTGGAACAGAInaGs;omeUs;InaGs;omeGs;InaAs;omeAs;InaCs;omeAs;InaGs; 10961 147 m01TACAT omeAs;InaTs;omeAs;InaCs;omeAs;InaT-Sup SMN1- TGGAACAGATInaTs;omeGs;InaGs;omeAs;InaAs;omeCs;InaAs;omeGs;InaAs; 10962 148 m01ACATA omeUs;InaAs;omeCs;InaAs;omeUs;InaA-Sup SMN1- TGTCCAGATTInaTs;omeGs;InaTs;omeCs;InaCs;omeAs;InaGs;omeAs;InaTs; 11367 149 m01CTCTT omeUs;InaCs;omeUs;InaCs;omeUs;InaT-Sup SMN1- GTCCAGATTCInaGs;omeUs;InaCs;omeCs;InaAs;omeGs;InaAs;omeUs;InaTs; 11368 150 m01TCTTG omeCs;InaTs;omeCs;InaTs;omeUs;InaG-Sup SMN1- TCCAGATTCTInaTs;omeCs;InaCs;omeAs;InaGs;omeAs;InaTs;omeUs;InaCs; 11369 151 m01CTTGA omeUs;InaCs;omeUs;InaTs;omeGs;InaA-Sup SMN1- CCAGATTCTCInaCs;omeCs;InaAs;omeGs;InaAs;omeUs;InaTs;omeCs;InaTs; 11370 152 m01TTGAT omeCs;InaTs;omeUs;InaGs;omeAs;InaT-Sup SMN1- CAGATTCTCTInaCs;omeAs;InaGs;omeAs;InaTs;omeUs;InaCs;omeUs;InaCs; 11371 153 m01TGATG omeUs;InaTs;omeGs;InaAs;omeUs;InaG-Sup SMN1- AGATTCTCTTInaAs;omeGs;InaAs;omeUs;InaTs;omeCs;InaTs;omeCs;InaTs; 11372 154 m01GATGA omeUs;InaGs;omeAs;InaTs;omeGs;InaA-Sup SMN1- GATTCTCTTGInaGs;omeAs;InaTs;omeUs;InaCs;omeUs;InaCs;omeUs;InaTs; 11373 155 m01ATGAT omeGs;InaAs;omeUs;InaGs;omeAs;InaT-Sup SMN1- GGAAGTATGTInaGs;omeGs;InaAs;omeAs;InaGs;omeUs;InaAs;omeUs;InaGs; 11400 156 m01TAATT omeUs;InaTs;omeAs;InaAs;omeUs;InaT-Sup SMN1- GAAGTATGTTInaGs;omeAs;InaAs;omeGs;InaTs;omeAs;InaTs;omeGs;InaTs; 11401 157 m01AATTT omeUs;InaAs;omeAs;InaTs;omeUs;InaT-Sup SMN1- AAGTATGTTAInaAs;omeAs;InaGs;omeUs;InaAs;omeUs;InaGs;omeUs;InaTs; 11402 158 m01ATTTC omeAs;InaAs;omeUs;InaTs;omeUs;InaC-Sup SMN1- AGTATGTTAAInaAs;omeGs;InaTs;omeAs;InaTs;omeGs;InaTs;omeUs;InaAs; 11403 159 m01TTTCA omeAs;InaTs;omeUs;InaTs;omeCs;InaA-Sup SMN1- GTATGTTAATInaGs;omeUs;InaAs;omeUs;InaGs;omeUs;InaTs;omeAs;InaAs; 11404 160 m01TTCAT omeUs;InaTs;omeUs;InaCs;omeAs;InaT-Sup SMN1- TATGTTAATTInaTs;omeAs;InaTs;omeGs;InaTs;omeUs;InaAs;omeAs;InaTs; 11405 161 m01TCATG omeUs;InaTs;omeCs;InaAs;omeUs;InaG-Sup SMN1- ATGTTAATTTInaAs;omeUs;InaGs;omeUs;InaTs;omeAs;InaAs;omeUs;InaTs; 11406 162 m01CATGG omeUs;InaCs;omeAs;InaTs;omeGs;InaG-Sup SMN1- TGAAATATTCInaTs;omeGs;InaAs;omeAs;InaAs;omeUs;InaAs;omeUs;InaTs; 10064 163 m01CTTAT omeCs;InaCs;omeUs;InaTs;omeAs;InaT-Sup SMN1- TATAGCCAGGInaTs;omeAs;InaTs;omeAs;InaGs;omeCs;InaCs;omeAs;InaGs; 10076 164 m01TCTAA omeGs;InaTs;omeCs;InaTs;omeAs;InaA-Sup SMN1- ATAGCCAGGTInaAs;omeUs;InaAs;omeGs;InaCs;omeCs;InaAs;omeGs;InaGs; 10077 165 m01CTAAA omeUs;InaCs;omeUs;InaAs;omeAs;InaA-Sup SMN1- AGGTCTAAAAInaAs;omeGs;InaGs;omeUs;InaCs;omeUs;InaAs;omeAs;InaAs; 10083 166 m01TTCAA omeAs;InaTs;omeUs;InaCs;omeAs;InaA-Sup SMN1- GTCTAAAATTInaGs;omeUs;InaCs;omeUs;InaAs;omeAs;InaAs;omeAs;InaTs; 10085 167 m01CAATG omeUs;InaCs;omeAs;InaAs;omeUs;InaG-Sup SMN1- TCTAAAATTCInaTs;omeCs;InaTs;omeAs;InaAs;omeAs;InaAs;omeUs;InaTs; 10086 168 m01AATGG omeCs;InaAs;omeAs;InaTs;omeGs;InaG-Sup SMN1- TAAAATTCAAInaTs;omeAs;InaAs;omeAs;InaAs;omeUs;InaTs;omeCs;InaAs; 10088 169 m01TGGCC omeAs;InaTs;omeGs;InaGs;omeCs;InaC-Sup SMN1- AAAATTCAATInaAs;omeAs;InaAs;omeAs;InaTs;omeUs;InaCs;omeAs;InaAs; 10089 170 m01GGCCC omeUs;InaGs;omeGs;InaCs;omeCs;InaC-Sup unc-232 CTACGCGTCGInaCs;dTs;InaAs;dCs;InaGs;dCs;InaGs;dTs;InaCs;dGs; 13095 m12 ACGGTInaAs;dCs;InaGs;dGs;InaT-Sup unc-232 CTACGCGTCGInaCs;omeUs;InaAs;omeCs;InaGs;omeCs;InaGs;omeUs;InaCs; 13095 m01 ACGGTomeGs;InaAs;omeCs;InaGs;omeGs;InaT-Sup unc-293 CCGATTCGCGInaCs;dCs;InaGs;dAs;InaTs;dTs;InaCs;dGs;InaCs;dGs; 13096 m12 CGTAAInaCs;dGs;InaTs;dAs;InaA-Sup unc-293 CCGATTCGCGInaCs;omeCs;InaGs;omeAs;InaTs;omeUs;InaCs;omeGs;InaCs; 13096 m01 CGTAAomeGs;InaCs;orneGs;InaTs;omeAs;InaA-Sup

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

What is claimed is:
 1. A composition comprising a cell and a singlestranded oligonucleotide, wherein the single stranded oligonucleotide isproduced by a process comprising: synthesizing a single strandedoligonucleotide that: (a) has a sequence 5′-X—Y—Z, wherein X is anynucleotide, Y is a nucleotide sequence of 6 nucleotides in length thatis not a seed sequence of a human microRNA, and Z is a nucleotidesequence of 1-8 nucleotides in length, (b) is 100% complementary with aPRC2-associated region of a human SMN gene, wherein the PRC2-associatedregion is a region of the SMN gene that has a sequence that occurs at ahigher frequency in a sequencing reaction of products of anRNA-immunoprecipitation assay that employs an antibody that targets Ezh2to immunoprecipitate RNA-associated PRC2 complexes from cells comprisingthe SMN gene compared to a control sequencing reaction of products of acontrol RNA-immunoprecipitation assay that employs a control antibody,and (c) is 8 to 15 nucleotides in length, wherein, during the synthesis,at least one nucleotide incorporated into the oligonucleotide comprisesa 2′ O-methyl and/or a 2′-fluoro, and a phosphorothioate internucleotidelinkage is incorporated between at least two nucleotides.
 2. Thecomposition of claim 1, wherein the oligonucleotide does not comprisethree or more consecutive guanosine nucleotides.
 3. The composition ofclaim 1, wherein the oligonucleotide does not comprise four or moreconsecutive guanosine nucleotides.
 4. The composition of claim 1,wherein at least one nucleotide of the oligonucleotide comprises a 2′O-methyl.
 5. The composition of claim 1, wherein each nucleotide of theoligonucleotide comprises a 2′ O-methyl.
 6. The composition of claim 1,wherein the oligonucleotide comprises at least one ribonucleotide, atleast one deoxyribonucleotide, or at least one bridged nucleotide. 7.The composition of claim 6, wherein the bridged nucleotide is a LNAnucleotide, a cEt nucleotide or a ENA modified nucleotide.
 8. Thecomposition of claim 1, wherein the nucleotides of the oligonucleotidecomprise alternating deoxyribonucleotides and2′-fluoro-deoxyribonucleotides.
 9. The composition of claim 1, whereinthe nucleotides of the oligonucleotide comprise alternatingdeoxyribonucleotides and 2′-O-methyl nucleotides.
 10. The composition ofclaim 1, wherein the nucleotides of the oligonucleotide comprisealternating deoxyribonucleotides and ENA nucleotide analogues.
 11. Thecomposition of claim 1, wherein the nucleotides of the oligonucleotidecomprise alternating deoxyribonucleotides and LNA nucleotides.
 12. Thecomposition of claim 8, wherein the 5′ nucleotide of the oligonucleotideis a deoxyribonucleotide.
 13. The composition of claim 1, wherein thenucleotides of the oligonucleotide comprise alternating LNA nucleotidesand 2′-O-methyl nucleotides.
 14. The composition of claim 13, whereinthe 5′ nucleotide of the oligonucleotide is a LNA nucleotide.
 15. Thecomposition of claim 1, wherein the nucleotides of the oligonucleotidecomprise deoxyribonucleotides flanked by at least one LNA nucleotide oneach of the 5′ and 3′ ends of the deoxyribonucleotides.
 16. Thecomposition of claim 1, further comprising phosphorothioateinternucleotide linkages between all nucleotides.
 17. The composition ofclaim 1, wherein the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group.
 18. The composition of claim 1,wherein the nucleotide at the 3′ position of the oligonucleotide has a3′ thiophosphate.
 19. The composition of claim 1, further comprising abiotin moiety conjugated to the 5′ nucleotide.
 20. A compositioncomprising a cell and a single stranded oligonucleotide, wherein thesingle stranded oligonucleotide is produced by a process comprising:synthesizing a single stranded oligonucleotide that: (a) is 100%complementary with a PRC2-associated region of a human SMN gene, whereinthe PRC2-associated region is a region of the SMN gene that has asequence that occurs at a higher frequency in a sequencing reaction ofproducts of an RNA-immunoprecipitation assay that employs an antibodythat targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexesfrom cells comprising the SMN gene compared to a control sequencingreaction of products of a control RNA-immunoprecipitation assay thatemploys a control antibody, (b) is 8 to 15 nucleotides in length and hasat least one of the following features i)-iv): i) a sequence that is5′X—Y—Z, wherein X is any nucleotide and wherein X is anchored at the 5′end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotidesin length that is not a human seed sequence of a microRNA, and Z is anucleotide sequence of 1-8 nucleotides in length; ii) a sequence thatdoes not comprise three or more consecutive guanosine nucleotides; iii)a sequence that is complementary to a PRC2-associated region thatencodes an RNA that forms a secondary structure comprising at least twosingle stranded loops; and/or iv) a sequence that has greater than 60%G-C content wherein, during the synthesis, at least one nucleotideincorporated into the oligonucleotide comprises a 2′ O-methyl and/or a2′-fluoro, and a phosphorothioate internucleotide linkage isincorporated between at least two nucleotides.
 21. The composition ofclaim 20, wherein the oligonucleotide has the sequence 5′X—Y-Z.
 22. Acomposition comprising a cell and a single stranded oligonucleotideconjugated to a carrier, wherein the single stranded oligonucleotide isproduced by a process comprising: synthesizing a single strandedoligonucleotide that: (a) has a sequence 5′-X—Y—Z, wherein X is anynucleotide, Y is a nucleotide sequence of 6 nucleotides in length thatis not a seed sequence of a human microRNA, and Z is a nucleotidesequence of 1 to 8 nucleotides in length, (b) is 100% complementary witha PRC2-associated region of a human SMN gene, wherein thePRC2-associated region is a region of the SMN gene that has a sequencethat occurs at a higher frequency in a sequencing reaction of productsof an RNA-immunoprecipitation assay that employs an antibody thattargets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes fromcells comprising the SMN gene compared to a control sequencing reactionof products of a control RNA-immunoprecipitation assay that employs acontrol antibody, and (c) is 8 to 15 nucleotides in length, wherein,during the synthesis, at least one nucleotide incorporated into theoligonucleotide comprises a 2′ O-methyl and/or a 2′-fluoro, and aphosphorothioate internucleotide linkage is incorporated between atleast two nucleotides.
 23. The composition of claim 22, wherein thecarrier is a peptide.
 24. The composition of claim 22, wherein thecarrier is a steroid.
 25. A pharmaceutical composition comprising acomposition of claim 22 and a pharmaceutically acceptable carrier.
 26. Akit comprising a container housing the composition of claim
 22. 27. Amethod of increasing expression of SMN1 or SMN2 in a human cell, themethod comprising: delivering a single stranded oligonucleotide into thecell, wherein the oligonucleotide does not induce substantial cleavageor degradation of the SMN1 or SMN2 mRNA in the cell and wherein thesingle stranded oligonucleotide is produced by a process comprising:synthesizing a single stranded oligonucleotide that: (a) has a sequence5′-X—Y—Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6nucleotides in length that is not a seed sequence of a human microRNA,and Z is a nucleotide sequence of 1 to 8 nucleotides in length, (b) is100% complementary with a PRC2-associated region of a human SMN gene,wherein the PRC2-associated region is a region of the SMN gene that hasa sequence that occurs at a higher frequency in a sequencing reaction ofproducts of an RNA-immunoprecipitation assay that employs an antibodythat targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexesfrom cells comprising the SMN gene compared to a control sequencingreaction of products of a control RNA-immunoprecipitation assay thatemploys a control antibody, and (c) is 8 to 15 nucleotides in length,wherein, during the synthesis, at least one nucleotide incorporated intothe oligonucleotide comprises a 2′ O-methyl and/or a 2′-fluoro, and aphosphorothioate internucleotide linkage is incorporated between atleast two nucleotides.
 28. The method of claim 27, wherein delivery ofthe single stranded oligonucleotide into the cell results in a level ofexpression of SMN1 or SMN2 mRNA that is at least 50% greater than alevel of expression of SMN1 or SMN2 mRNA in a control cell that does notcomprise the single stranded oligonucleotide.
 29. A method of increasingexpression of SMN2 messenger RNA (mRNA) in a human cell, the methodcomprising: delivering to the cell a first single strandedoligonucleotide complementary with at least 8 consecutive nucleotides ofa PRC2-associated region of human SMN2 and a second single strandedoligonucleotide complementary with at least 8 consecutive nucleotides ofa splice control sequence of a precursor mRNA of human SMN2, in amountssufficient to increase expression of a mature mRNA of SMN2 thatcomprises exon 7 in the cell, wherein the first and secondoligonucleotides do not induce substantial cleavage or degradation ofSMN2 mRNA in the cell, wherein at least one nucleotide of the firstsingle stranded oligonucleotide comprises a 2′ O-methyl and/or a2′-fluoro, and the first single stranded oligonucleotide comprises aphosphorothioate internucleotide linkage between at least twonucleotides.
 30. The method of claim 29, wherein the first singlestranded oligonucleotide is covalently linked to the second singlestranded oligonucleotide through a linker.
 31. A composition comprising:a first single stranded oligonucleotide produced by a process comprisingsynthesizing a first single stranded oligonucleotide that iscomplementary with at least 8 consecutive nucleotides of aPRC2-associated region of a human SMN2 gene, wherein the PRC2-associatedregion is a region of the SMN2 gene that has a sequence that occurs at ahigher frequency in a sequencing reaction of products of anRNA-immunoprecipitation assay that employs an antibody that targets Ezh2to immunoprecipitate RNA-associated PRC2 complexes from cells comprisingthe SMN2 gene compared to a control sequencing reaction of products of acontrol RNA-immunoprecipitation assay that employs a control antibody,and a second single stranded oligonucleotide complementary with at least9 consecutive nucleotides of a splice control sequence of a precursormRNA encoded by the SMN2 gene, wherein, during the synthesis, at leastone nucleotide incorporated into the first single strandedoligonucleotide comprises a 2′ O-methyl and/or a 2′-fluoro, and aphosphorothioate internucleotide linkage is incorporated between atleast two nucleotides.
 32. The composition of claim 31, wherein thefirst single stranded oligonucleotide is covalently linked to the secondsingle stranded oligonucleotide through a linker.